Fusion proteins comprising pdgf and vegf binding portions and methods of using thereof

ABSTRACT

The present provides fusion proteins comprising PDGF and VEGF binding portions, and recombinant viral particles encoding the fusion proteins. Compositions comprising the fusion proteins and viral particles as well as methods of using the same are also provided.

CROSS REFERENCE TO RELATED APPLICATION

This application claims the benefit under 35 §USC 119(e) of priorco-pending U.S. Provisional Patent Application No. 61/780,914, filedMar. 13, 2013, the disclosure of which is hereby incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to fusion proteins that inhibit the PDGFpathway and the VEGF pathway, compositions of these fusion proteins aswell as methods for producing and using the same.

BACKGROUND OF THE INVENTION

Formation of new blood vessels, caused by the overproduction of growthfactors such as vascular endothelial growth factor (VEGF), is a keycomponent of diseases like tumor growth, age-related maculardegeneration (AMD) and proliferative diabetic retinopathy (PDR)(Connolly et al., J Clin Invest., 1989, 84(5):1470-8; Ferrara et al.,Biochem Biophys Res Commun., 1989, 161(2):851-9; and Ferrara et al.,Nat. Med., 1998, 4(3):336-40). Wet AMD is the most severe form of AMDdisease that is characterized by abnormal neovascularization beneath theretina and often leads to permanent vision loss. Blocking of VEGF withantibodies, soluble VEGF receptors, or inhibition of VEGF receptortyrosine kinase activity are strategies that have shown promisingpreclinical and clinical results in the suppression of retinalneovascularization (Aiello et al., PNAS, 1995, 92:10457-10461 andWillet, et al., Nat. Med., 2004, 10:145-147). However, recent clinicaldata shows that new vascular tissue typically does not regress with VEGFinhibition alone, because pericytes, which interact with endothelialcells and contribute to the establishment of the blood-retinal barrier,provide survival signals to neovascular endothelial cells and hence makethem resistant to VEGF withdrawal (Benjamin et al., Development, 1998,125(9)1591-8 and Patel S., Retina, 2009, 29(6 Suppl):545-8).Furthermore, platelet-derived growth factor isoform B (PDGF-B) and PDGFreceptor-beta (PDGFRβ), found in proliferative retinal membranes, haveimportant roles in recruitment of pericytes for stabilization of thedeveloping vasculature (Robbins et al., Invest Opth Vis Sci., 1994,35(10):3649-63; Lindahl et al., Development, 1997, 124:3943-3953; andHellström et al., Development, 1999, 126:3047-3055).

The VEGF binding function of VEGFR1 (Flt-1) has been mapped to thesecond extracellular domain (ECD) (Davis-Smyth et al., EMBO J., 1996,15:4919-4927; Barleon et al., J Biol. Chem., 1997, 272:10382-10388;Wiesmann et al., Cell, 1997, 91:695-704; and Davis-Smyth et al., J Biol.Chem., 1998, 273:3216-3222). A naturally occurring alternatively splicedform of high affinity VEGF-binding receptor, soluble VEGFR1 (sFlt1),exists as a secreted protein that functions primarily as a decoyreceptor (Shibuya et al., Oncogene, 1990, 5:519-524 and Kendall et al.,PNAS, 1993, 90:10705-10709). A soluble receptor, VEGF-Trap, engineeredfor therapeutic use, has the second domain of VEGFR1 fused to the thirddomain of VEGFR2 (KDR) and to human IgG1 Fc region (Holash et al. 2002).An extracellular region of PDGFRβ was previously shown to antagonizePDGF-B stimulated responses (Duan et al., J Biol Chem, 1991, 266(1)413-8and Ueno et al., Science, 1991, 252(5007):844-8). Studies with PDGFRβ-Fcchimera demonstrated that human PDGFRβ ECDs 1 to 3 are sufficient forhigh-affinity PDGF-B ligand binding (Heidaran et al., FASEB J., 1995,9(1):140-5 and Lokker et al., J Biol Chem, 1997, 272(52):33037-44). Aneffect of predimerization on high-affinity PDGF-B ligand binding wasalso described when PDGFRβ ECDs 1 to 3 were fused to glutathioneS-transferase (GST) domain (Leppänen et al., Biochemistry, 2000,39(9):2370-5).

Current eye treatments require monthly intravitreal injections for yearsby a retinal specialist. Therefore, there is a need for improvedtherapeutic agents and an approach to deliver such therapeutic agents tosites such as the eye.

BRIEF SUMMARY OF THE INVENTION

The invention provided herein discloses, inter alia, fusion proteinsthat inhibit the PDGF pathway and the VEGF pathway, compositionscomprising these fusion proteins and compositions comprising viralparticles comprising a nucleic acid encoding the fusion protein, as wellas methods for producing and using these fusion proteins and viralparticles for the treatment or prevention of a disease such as an oculardisease, an inflammatory disease, an autoimmune disease, or cancer.

Accordingly, in one aspect, the invention provides a fusion proteincomprising (a) an extracellular portion of a PDGF receptor, (b) anextracellular portion of a VEGF receptor, and (c) a multimerizationdomain, wherein the fusion proteins binds to a PDGF and a VEGF. In someembodiments, the fusion protein is arranged from N-terminus toC-terminus in the following order: (a), (b) and (c). In someembodiments, the PDGF receptor is a PDGFRβ. In some embodiments herein,the extracellular portion of the PDGFR comprises the Ig-like domainsD1-D3 of the PDGFR. In some embodiments herein, the extracellularportion of the PDGFR comprises the Ig-like domains D1-D4 of the PDGFR.In some embodiments herein, the extracellular portion of the PDGFRcomprises the Ig-like domains D1-D5 of the PDGFR. In some embodimentsherein, the extracellular portion of the PDGFR comprises the amino acidsequence SEQ ID NO:1, 2, or 3, or an amino acid sequence having at least85% identity to SEQ ID NO:1, 2, or 3. In some embodiments herein, theextracellular portion of the VEGF receptor comprises an Ig-like domainD2 of a VEGF receptor. In some embodiments herein, the extracellularportion of the VEGF receptor comprises an Ig-like domain D2 of a VEGFR1(FLT-1). In some embodiments herein, the extracellular portion of theVEGF receptor comprises an Ig-like domain D2 of a VEGFR1 (FLT-1) and anIg-like domain D3 of a VEGFR2. In some embodiments herein, theextracellular portion of the VEGF receptor comprises the Ig-like domainsD1-D3 of a VEGFR1 (FLT-1). In some embodiments herein, the extracellularportion of the VEGF receptor comprises the amino acid sequence of SEQ IDNO:4 or 5, or an amino acid sequence having at least 85% identity to SEQID NO:4 or 5. In some embodiments herein, the fusion protein furthercomprises a linker peptide between the extracellular portion of the PDGFreceptor and the extracellular portion of the VEGF receptor, and/or apeptide linker between the extracellular portion of the VEGF receptorand the multimerization domain. In a further embodiment, the peptidelinker comprises the amino acid sequence selected from the groupconsisting of Gly₉, Glu₉, Ser₉, Gly₅ Cys-Pro₂-Cys, (Gly₄-Ser)₃,Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn,Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn,Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys, andGly₉-Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn. In someembodiments herein, the multimerization domain is a Fc region of anantibody. In a further embodiment, the Fc region comprises a CH3 regionof IgG1, IgG2, IgG3, or IgG4, or a CH2 and a CH3 region of IgG1, IgG2,IgG3, or IgG4. In some embodiments herein, the Fc region comprises theamino acid sequence of SEQ ID NO:6, or an amino acid sequence having atleast 85% identity to SEQ ID NO:6. In some embodiments herein, thefusion protein comprises the amino acid sequence of SEQ ID NO:13 or 15,or an amino acid sequence having at least 85% identity to SEQ ID NO:13or 15. In some embodiments herein, the fusion protein is in a multimericform. In some of the embodiments herein, the fusion protein is in adimeric form.

In one aspect, the invention provides a fusion protein produced byculturing a host cell comprising a nucleic acid encoding any of thefusion proteins disclosed herein under a condition that produces thefusion protein, and recovering the fusion protein produced by the hostcell.

In another aspect, the invention provides a dimeric fusion proteincomprising two fusion proteins, wherein each fusion protein comprisesany of the fusion proteins disclosed herein.

In yet another aspect, the invention provides a composition comprisingany of the fusion proteins disclosed herein and a pharmaceuticallyacceptable carrier.

In still another aspect, the invention provides a nucleic acid encodingany of the fusion proteins disclosed herein.

In some aspects, the invention also provides a host cell comprising anucleotide sequence encoding any of the fusion proteins disclosedherein.

In some aspects, the invention provides a method of producing a fusionprotein, comprising culturing a host cell comprising a nucleic acidencoding any of the fusion proteins disclosed herein under a conditionthat produces the fusion protein, and recovering the fusion proteinproduced by the host cell. In further embodiments, the host cell is amammalian cell.

In another aspect, the invention provides a method of delivering afusion protein to a subject comprising administering an effective amountof any of the fusion proteins disclosed herein to the subject. In someembodiments, the subject has macular degeneration or proliferativediabetic retinopathy. In a further embodiment, the macular degenerationis wet age-related macular degeneration or dry age-related maculardegeneration. In some embodiments herein, the fusion protein isadministered by intravitreal injection to the subject. In someembodiments, the subject has cancer. In some embodiments, the subjecthas rheumatoid arthritis, osteoarthritis, or asthma. In someembodiments, the subject has uveitis or corneal neovascularization.

In some aspects, the invention provides a vector comprising a nucleotidesequence encoding any of the fusion proteins disclosed herein. In someembodiments, the vector is a viral vector. In a further embodiment, theviral vector is a recombinant adeno-associated virus vector (rAAV). Infurther embodiments, the rAAV vector comprises an ITR of AAV1, AAV2,AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, or AAVrh10.

In one aspect, the invention also provides an rAAV particle comprising anucleic acid encoding any of the fusion proteins disclosed herein. Insome embodiments, the rAAV particle comprises capsid proteins of AAV1,AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAVrh8, or AAVrh10. Insome embodiments, the nucleic acid comprises an ITR from a serotypedifferent from the serotype of the capsid. In a further embodiment, theITR is an ITR of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9,AAVrh8, or AAVrh10.

In yet another aspect, the invention provides a method of producing anrAAV particle, comprising (a) culturing a host cell under a conditionthat rAAV particles are produced, wherein the host cell comprises (i)one or more AAV package genes, wherein each said AAV packaging geneencodes an AAV replication or encapsidation protein; (ii) an rAAVpro-vector comprising a nucleotide encoding any of the fusion proteinsdisclosed herein flanked by at least one AAV ITR, and (iii) an AAVhelper function; and (b) recovering the rAAV particles produced by thehost cell. In a further embodiment, the rAAV particles are purified.

In still another aspect, the invention provides a method of delivering aviral vector to a subject, comprising administering any of the rAAVparticles disclosed herein to the subject, wherein the fusion proteinencoded by the rAAV particle is expressed in the subject. In someembodiments, the subject has macular degeneration or proliferativediabetic retinopathy. In a further embodiment, the macular degenerationis wet age-related macular degeneration or dry age-related maculardegeneration. In some embodiments herein, the rAAV particle isadministered by intravitreal injection to the subject. In someembodiments, the subject has cancer. In some embodiments, the subjecthas rheumatoid arthritis, osteoarthritis, or asthma. In someembodiments, the subject has uveitis or corneal neovascularization.

The specification is considered to be sufficient to enable one skilledin the art to practice the invention. Various modifications of theinvention in addition to those shown and described herein will becomeapparent to those skilled in the art from the foregoing description andfall within the scope of the appended claims. All publications, patents,and patent applications cited herein are hereby incorporated byreference in their entirety for all purposes.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows generation of truncated PDGFR-β soluble receptors. A)Schematic of the PDGFR-β IgG1 Fc-coupled dimerizing forms,PDGFR(D1-D5)9G-Fc, PDGFR(D1-D2)9G-Fc, and PDGFR(D1-D3)9G-Fc as well asthe PDGFR-β monomeric receptor forms, PDGFR(D1-D4) and PDGFR(D1-D5).White blocks indicate PDGFR-β sequences, including the extracellulardomains and the signal peptide (sp). Diagonal shaded blocks represent9Gly linker and dark dotted blocks represent domains CH2 and CH3 ofhuman IgG1 Fc region. B) Western blots of monomeric sPDGFR-β and IgG1Fc-coupled dimerizing soluble receptor forms under reducing (left panel)and non-reducing (right panel) conditions. Protein was detected withanti-PDGFR-β antibody.

FIG. 2 shows a volumetric PDGF BB binding assay of truncated PDGFR-βsoluble receptors. A) Monomeric PDGFR-β soluble receptor formsPDGFR(D1-D4) and PDGFR(D1-D5) as compared to the full-size IgG1Fc-coupled dimerizing form PDGFR(D1-D5)9G-Fc. B) Dimeric IgG1 Fc-coupledsPDGFR-β soluble receptor forms PDGFR(D1-D2)9G-Fc and PDGFR(D1-D3)9G-Fcas compared to the IgG1 Fc-coupled dimerizing form PDGFR(D1-D5)9G-Fc.Increasing volumes (μl) of conditioned media (CM) containing solublereceptors (x axis) from representative transfections were incubatedovernight with human PDGF BB ligand and the amount of unbound ligand (yaxis) was measured by ELISA. Data expressed as mean±SD (n=3); allreceptors were significantly different in PDGF binding affinities by2-way ANOVA; Bonferroni Test; ***P<0.001.

FIG. 3 is a schematic of VEGFR1/PDGFR-β and PDGFR-β/VEGFR1 hybridproteins, Hybrids 1 to 4, and their parental constructsPDGFR(D1-D5)9G-Fc, PDGFR(D1-D3)9G-Fc and sFLT01. White blocks indicatePDGFR-β sequences, grey blocks indicate VEGFR1 (Flt-1) sequences,including their extracellular domains and signal peptides (sp). Diagonalshaded blocks represent 9Gly or 9Ser linkers and dark dotted blocksrepresent domains CH2 and CH3 of the human IgG1 Fc region.

FIG. 4 is a Western blot of VEGFR1/PDGFR-β and PDGFR-β/VEGFR1 hybridproteins, Hybrids 1 to 4, as compared to full-size IgG1 Fc-coupleddimerizing form PDGFR(D1-D5)9G-Fc (shown as (D1-D5)9G-Fc) under reducing(left panel) and non-reducing (right panel) conditions. Protein wasdetected with anti-PDGFR-β antibody and anti-Flt-1 antibody. Samplescontaining Hybrids 3 and 4 were duplicates from individualtransfections.

FIG. 5 shows graphs demonstrating inhibition of VEGF-induced and VEGF+PDGF-β-induced proliferation of human umbilical vein endothelial cells(HUVECs) by hybrid protein, Hybrids 1 to 4. A) HUVEC proliferation assaywith VEGF only: Inhibitory effect of 5 μl conditioned media (CM)containing soluble receptors on HUVEC proliferation was compared in thepresence of VEGF (10 ng/ml) only. B) HUVEC competitive proliferationassay in the presence of VEGF (10 ng/ml) and PDGF (20 ng/ml): Inhibitoryeffect of 5 μl CM containing soluble receptors on HUVEC proliferationwas compared in the presence of both ligands, VEGF and PDGF. Samplesfrom three independent transfections (n=3) were evaluated in one assay.Data expressed as mean±SD. One-way ANOVA; Tukey's Test; ***p<0.001 fordifference between positive control VEGF+ alone or VEGF+ in combinationwith PDGF BB+ versus other samples. Control=EGFP CM; VEGF+=EGFP CM+10ng/ml VEGF; BB+Only=EGFP CM+20 ng/ml; VEGF+BB+=EGFP CM+10 ng/ml VEGF+20ng/ml PDGF BB.

FIG. 6 shows volumetric binding assays of hybrid proteins. A) PDGF BBvolumetric binding assay of Hybrid proteins 1 to 4 as compared toPDGFR(D1-D5)9G-Fc. B) VEGF volumetric binding assay of Hybrid proteins 1to 4 as compared to sFLT01. Increasing conditioned media volumescontaining soluble receptors (x axis) from representative transfectionswere incubated overnight with either human PDGF BB or VEGF ligands andthe amount of unbound ligand (y axis) was measured by ELISA intriplicates.

FIG. 7 shows competitive VEGF and PDGF cell-free binding assays ofhybrid proteins. A) Comparison of Hybrid 3 (Hyb#3), Hybrid 4 (Hyb #4),and PDGFR(D1-D3)9G-Fc in increasing conditioned media volumes (x-axis)and the amount of the unbound PDGF ligand (y axis) as measured by PDGFBB ELISA. B) Comparison of Hybrid 3 (Hyb#3), Hybrid 4 (Hyb #4), andsFltT01 in increasing conditioned media volumes (x-axis) and the amountof the unbound VEGF ligand (y axis) as measured by VEGF ELISA.

FIG. 8 is a graph showing in vivo efficacy of AAV2.Hybrid 4 intravitrealdelivery in a mouse choroidal neovascularization (CNV) laser model.Number of burns without neovascularization (NV) in the AAV2.Hybrid 4(shown as Hybrid-4), AAV2.sFLT02 (shown as sFLT02), AAV2.PDGFR (shown asPDGFR) treated (left) eye was compared to the untreated contralateral(right; Naive) eye. Data from both eyes (n=20 eyes per treatment) wasexpressed as percentage of burns without CNV. The PDGFR portion used forconstruction of AAV2.PDGFR was PDGFR(D1-D3)9G-Fc.

DETAILED DESCRIPTION

The present invention provides, inter alia, fusion proteins, andcompositions thereof, that inhibit the plasma-derived growth factor(PDGF) signaling pathway and the vascular endothelial growth factor(VEGF) signaling pathway. A fusion protein of the invention as describedherein comprises an extracellular portion of a PDGF receptor (PDGFR), anextracellular portion of a VEGF receptor (VEGFR), and a multimerizationdomain, wherein the fusion protein binds to a PDGF and a VEGF forinhibition of PDGF activity and VEGF activity, respectively. Alsoprovided herein are methods for production of the fusion proteins,methods of delivery of the fusion proteins, and methods of using thefusion proteins in the treatment of ocular diseases, autoimmunediseases, inflammatory diseases, and/or cancer.

I. General Techniques

The techniques and procedures described or referenced herein aregenerally well understood and commonly employed using conventionalmethodology by those skilled in the art, such as, for example, thewidely utilized methodologies described in Molecular Cloning: ALaboratory Manual (Sambrook et al., 4^(th) ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 2012); Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., 2003); the series Methodsin Enzymology (Academic Press, Inc.); PCR 2: A Practical Approach (M. J.MacPherson, B. D. Hames and G. R. Taylor eds., 1995); Antibodies, ALaboratory Manual (Harlow and Lane, eds., 1988); Culture of AnimalCells: A Manual of Basic Technique and Specialized Applications (R. I.Freshney, 6^(th) ed., J. Wiley and Sons, 2010); OligonucleotideSynthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, HumanaPress; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., AcademicPress, 1998); Introduction to Cell and Tissue Culture (J. P. Mather andP. E. Roberts, Plenum Press, 1998); Cell and Tissue Culture: LaboratoryProcedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., J. Wileyand Sons, 1993-8); Handbook of Experimental Immunology (D. M. Weir andC. C. Blackwell, eds., 1996); Gene Transfer Vectors for Mammalian Cells(J. M. Miller and M. P. Calos, eds., 1987); PCR: The Polymerase ChainReaction, (Mullis et al., eds., 1994); Current Protocols in Immunology(J. E. Coligan et al., eds., 1991); Short Protocols in Molecular Biology(Ausubel et al., eds., J. Wiley and Sons, 2002); Immunobiology (C. A.Janeway et al., 2004); Antibodies (P. Finch, 1997); Antibodies: APractical Approach (D. Catty., ed., IRL Press, 1988-1989); MonoclonalAntibodies: A Practical Approach (P. Shepherd and C. Dean, eds., OxfordUniversity Press, 2000); Using Antibodies: A Laboratory Manual (E.Harlow and D. Lane, Cold Spring Harbor Laboratory Press, 1999); TheAntibodies (M. Zanetti and J. D. Capra, eds., Harwood AcademicPublishers, 1995); and Cancer: Principles and Practice of Oncology (V.T. DeVita et al., eds., J. B. Lippincott Company, 2011).

II. Definitions

A “vector,” as used herein, refers to a recombinant plasmid or virusthat comprises a nucleic acid to be delivered into a host cell, eitherin vitro or in vivo.

The term “polynucleotide” or “nucleic acid” as used herein refers to apolymeric form of nucleotides of any length, either ribonucleotides ordeoxyribonucleotides. Thus, this term includes, but is not limited to,single-, double- or multi-stranded DNA or RNA, genomic DNA, cDNA,DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases, orother natural, chemically or biochemically modified, non-natural, orderivatized nucleotide bases. The backbone of the polynucleotide cancomprise sugars and phosphate groups (as may typically be found in RNAor DNA), or modified or substituted sugar or phosphate groups.Alternatively, the backbone of the polynucleotide can comprise a polymerof synthetic subunits such as phosphoramidates and thus can be aoligodeoxynucleoside phosphoramidate (P—NH₂) or a mixedphosphoramidate-phosphodiester oligomer. In addition, a double-strandedpolynucleotide can be obtained from the single stranded polynucleotideproduct of chemical synthesis either by synthesizing the complementarystrand and annealing the strands under appropriate conditions, or bysynthesizing the complementary strand de novo using a DNA polymerasewith an appropriate primer.

A “recombinant viral vector” refers to a recombinant polynucleotidevector comprising one or more heterologous sequences (i.e., nucleic acidsequence not of viral origin). In the case of recombinant AAV vectors,the recombinant nucleic acid is flanked by at least one, preferably two,inverted terminal repeat sequences (ITRs).

A “recombinant AAV vector (rAAV vector)” refers to a polynucleotidevector comprising one or more heterologous sequences (i.e., nucleic acidsequence not of AAV origin) that are flanked by at least one, preferablytwo, AAV inverted terminal repeat sequences (ITRs). Such rAAV vectorscan be replicated and packaged into infectious viral particles whenpresent in a host cell that has been infected with a suitable helpervirus (or that is expressing suitable helper functions) and that isexpressing AAV rep and cap gene products (i.e. AAV Rep and Capproteins). When a rAAV vector is incorporated into a largerpolynucleotide (e.g. in a chromosome or in another vector such as aplasmid used for cloning or transfection), then the rAAV vector may bereferred to as a “pro-vector” which can be “rescued” by replication andencapsidation in the presence of AAV packaging functions and suitablehelper functions. An rAAV can be in any of a number of forms, including,but not limited to, plasmids, linear artificial chromosomes, complexedwith lips, encapsulated within liposomes, and, most preferable,encapsidated in a viral particle, particularly AAV. A rAAV vector can bepackaged into an AAV virus capsid to generate a “recombinantadeno-associated virus particle (rAAV particle)”.

“Heterologous” means derived from a genotypically distinct entity fromthat of the rest of the entity to which it is compared or into which itis introduced or incorporated. For example, a polynucleotide introducedby genetic engineering techniques into a different cell type is aheterologous polynucleotide (and, when expressed, can encode aheterologous polypeptide). Similarly, a cellular sequence (e.g., a geneor portion thereof) that is incorporated into a viral vector, is aheterologous nucleotide sequence with respect to the vector.

An “inverted terminal repeat” or “ITR” sequence is a term wellunderstood in the art and refers to relatively short sequences found atthe termini of viral genomes which are in opposite orientation.

An “AAV inverted terminal repeat (ITR)” sequence, a term well-understoodin the art, is an approximately 145-nucleotide sequence that is presentat both termini of the native single-stranded AAV genome. The outermost125 nucleotides of the ITR can be present in either of two alternativeorientations, leading to heterogeneity between different AAV genomes andbetween the two ends of a single AAV genome. The outermost 125nucleotides also contains several shorter regions ofself-complementarity, allowing intrastrand base-pairing to occur withinthis portion of the ITR.

A “helper virus” for AAV refers to a virus that allows AAV (which is adefective parvovirus) to be replicated and packaged by a host cell. Anumber of such helper viruses have been identified, includingadenoviruses, herpesviruses and poxviruses such as vaccinia. Theadenoviruses encompass a number of different subgroups, althoughAdenovirus type 5 of subgroup C (Ad5) is most commonly used. Numerousadenoviruses of human, non-human mammalian and avian origin are knownand are available from depositories such as the ATCC. Viruses of theherpes family, which are also available from depositories such as ATCC,include, for example, herpes simplex viruses (HSV), Epstein-Barr viruses(EBV), cytomegaloviruses (CMV) and pseudorabies viruses (PRV).

A “fusion protein” refers to a protein having two or more portionscovalently linked together, where each of the portions is derived fromdifferent proteins.

“Percent (%) sequence identity” with respect to a reference polypeptideor nucleic acid sequence is defined as the percentage of amino acidresidues or nucleotides in a candidate sequence that are identical withthe amino acid residues or nucleotides in the reference polypeptide ornucleic acid sequence, after aligning the sequences and introducinggaps, if necessary, to achieve the maximum percent sequence identity,and not considering any conservative substitutions as part of thesequence identity. Alignment for purposes of determining percent aminoacid or nucleic acid sequence identity can be achieved in various waysthat are within the skill in the art, for instance, using publiclyavailable computer software programs, for example, those described inCurrent Protocols in Molecular Biology (Ausubel et al., eds., 1987),Supp. 30, section 7.7.18, Table 7.7.1, and including BLAST, BLAST-2,ALIGN or Megalign (DNASTAR) software. A preferred alignment program isALIGN Plus (Scientific and Educational Software, Pennsylvania). Thoseskilled in the art can determine appropriate parameters for measuringalignment, including any algorithms needed to achieve maximal alignmentover the full length of the sequences being compared. For purposesherein, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows: 100 times thefraction X/Y, where X is the number of amino acid residues scored asidentical matches by the sequence alignment program in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. For purposes herein, the % nucleic acidsequence identity of a given nucleic acid sequence C to, with, oragainst a given nucleic acid sequence D (which can alternatively bephrased as a given nucleic acid sequence C that has or comprises acertain % nucleic acid sequence identity to, with, or against a givennucleic acid sequence D) is calculated as follows: 100 times thefraction W/Z, where W is the number of nucleotides scored as identicalmatches by the sequence alignment program in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C.

An “isolated” molecule (e.g., nucleic acid or protein) or cell means ithas been identified and separated and/or recovered from a component ofits natural environment.

An “effective amount” is an amount sufficient to effect beneficial ordesired results, including clinical results. An effective amount can beadministered in one or more administrations. In terms of a diseasestate, an effective amount is an amount sufficient to ameliorate,stabilize, or delay development of a disease.

An “individual” or “subject” is a mammal. Mammals include, but are notlimited to, domesticated animals (e.g., cows, sheep, cats, dogs, andhorses), primates (e.g., humans and non-human primates such as monkeys),rabbits, and rodents (e.g., mice and rats). In certain embodiments, theindividual or subject is a human.

As used herein, “treatment” is an approach for obtaining beneficial ordesired clinical results. For purposes of this invention, beneficial ordesired clinical results include, but are not limited to, alleviation ofsymptoms, diminishment of extent of disease, stabilized (i.e., notworsening) state of disease, preventing spread (i.e., metastasis) ofdisease, delay or slowing of disease progression, amelioration orpalliation of the disease state, and remission (whether partial ortotal), whether detectable or undetectable. “Treatment” can also meanprolonging survival as compared to expected survival if not receivingtreatment.

Reference to “about” a value or parameter herein includes (anddescribes) embodiments that are directed to that value or parameter perse. For example, description referring to “about X” includes descriptionof “X.”

As used herein, the singular form of the articles “a,” “an,” and “the”includes plural references unless indicated otherwise. For example, thephrase “a rAAV particle” includes one or more rAAV particles.

It is understood that aspects and embodiments of the invention describedherein include “comprising,” “consisting,” and/or “consistingessentially of” aspects and embodiments.

III. Fusion Proteins and Fusion Protein Components

Plasma-Derived Growth Factor (PDGF) Receptor

Plasma-derived growth factors (PDGFs) are involved in many biologicalactivities and have been implicated in a number of diseases such asatherosclerosis, glomerulonephritis, vascular restenosis followingangioplasty, and cancer. There are at least four members of theplasma-derived growth factor (PDGF) family of proteins that regulate thePDGF signaling pathway, specifically PDGF-A, PDGF-B, PDGF-C, and PDGF-D.These four PDGFs assemble into disulfide-linked dimers via homo- orheterodimerization. At least five different dimeric isoforms of PDGFhave been described to date and include PDGF-AA, PDGF-BB, PDGF-CC,PDGF-DD, and PDGF-AB, all of which bind to PDGF receptors (PDGFRs) toactivate the PDGF signaling pathway. There are at least two identifiedPDGFRs, PDGFR-α and PDGFR-β. Each PDGFR has an extracellular region, atransmembrane domain, and an intracellular region having intracellulartyrosine kinase activity. PDGFRs can dimerize to form the homodimersPDGFR-α/PDGFR-α or PDGFR-β/PDGFR-β and the heterodimer PDGFR-α/PDGFR-β.Each of these PDGFR dimer forms recognize different dimeric isoforms ofPDGF. For example, PDGFR-α/PDGFR-α recognizes PDGF-AA, AB, BB and CCligands, PDGFR-α/PDGFR-β recognizes PDGF-AB, BB, CC, and DD, andPDGFR-β/PDGFR-β recognizes PDGF-BB and DD. Deletion mutagenesis of thePDGF-AA and -BB binding sites have been mapped to amino acids 1-314 ofPDGFR-α while the PDGF-BB binding sites have been mapped to amino acids1-315 of PDGFR-β. The extracellular region of these PDGFRs, whichmediate binding to PDGFs contain five immunoglobulin (Ig)-like domains,each ranging from about 88 to about 114 amino acids in length. SeeLokker et al., J Biol. Chem., 1997, 272(52):33037-44, Miyazawa et al., JBiol. Chem., 1998, 273(39):25495-502; and Mahadevan et al., J Biol.Chem., 1995, 270(46):27595-600, which are incorporated herein byreference their entirety.

The present invention provides an extracellular portion of a PDGFreceptor that can be a component of any fusion protein disclosed herein.Accordingly, in one aspect, the invention provides for an extracellularportion of a PDGFR that includes, but is not limited to, PDGFR-α andPDGFR-β. In some of the embodiments herein, the PDGFR is from a mammal,such as a human. There are five Ig-like domains numbered 1, 2, 3, 4, and5 starting from the N-terminus to the C-terminus of a PDGFRextracellular region. As used herein the terms “extracellular portion ofa PDGFR” refers to one or more of the five Ig-like domains in the PDGFRextracellular region. For example, “an extracellular portion of a PDGFR”refers to one or more of any of the five Ig-like domains found in theextracellular region of a PDGFR such as Ig-like domain D1, Ig-likedomain D2, Ig-like domain D3, Ig-like domain D4, or Ig-like domain D5.As used herein, terms such as “Ig-like domain D1” or “extracellulardomain (ECD) 1” of a PDGFR specifically refers to the first Ig-likedomain found at the N-terminus of the extracellular region of PDGFR,“Ig-like domain D2” or “ECD 1” of a PDGFR specifically refers to thesecond Ig-like domain from the N-terminus of the extracellular region ofPDGFR, and so forth. In any of the aspects herein, an extracellularportion of a PDGFR comprises at least one Ig-like domain of one or morePDGFRs selected from the group consisting of PDGFR-α and PDGFR-β. Insome aspects, an extracellular portion of a PDGFR comprises at least 1,2, 3, 4, but no more than 5 Ig-like domains of a PDGFR (e.g., PDGFR-β).In some aspects, an extracellular portion of a PDGFR comprises 1 to 5, 1to 4, 1 to 3, or 1 to 2 Ig-like domains of a PDGFR (e.g., PDGFR-β). Forexample, an extracellular portion of a PDGFR can comprise an Ig-likedomain D2 of a PDGFR. In another example, an extracellular portion of aPDGFR can comprise of Ig-like domains D1 to D2 of a PDGFR (e.g.,PDGFR-β). In yet another example, an extracellular portion of a PDGFRcan comprise the Ig-like domains D1 to D3, the Ig-like domains D1 to D4,or the Ig-like domains D1 to D5 of a PDGFR (e.g., PDGFR-β).

An extracellular portion comprising any combination of the five Ig-likedomains of each PDGFR are contemplated herein. Accordingly, in oneaspect, the present invention provides an extracellular portion of aPDGFR comprising at least one Ig-like domain of two PDGFRs. In someembodiments, an extracellular portion of a PDGFR comprises at least oneIg-like domain from two PDGFRs selected from the group consisting ofPDGFR-α and PDGFR-β. For example, a fusion protein as described hereincan comprise an extracellular portion of a PDGFR comprising at least oneIg-like domain of PDGFR-α and at least one Ig-like domain of PDGFR-β. Insome aspects, an extracellular portion of a PDGFR comprises at least 1,2, 3, 4, 5, 6, 7, 8, 9, but no more than 10 Ig-like domains of at leasttwo or more PDGFRs. In a further aspect, an extracellular portion of aPDGFR comprises 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4,1 to 3, or 1 to 2 Ig-like domains of at least two or more PDGFRs. For afurther description of Ig-like domains that can be used as part of anextracellular portion of a PDGFR, see U.S. Pat. No. 5,686,572,WO2006113277, and Lokker et al., J Biol. Chem. 1997, 272(52):33037-44,all of which are incorporated herein by reference in their entirety.

In some aspects, an extracellular portion of a PDGFR comprises the aminoacid sequence selected from the group consisting of SEQ ID NOs:1-3. Forexample, an extracellular portion of a PDGFR comprising the amino acidsequence of SEQ ID NO:1, SEQ ID NO:2, or SEQ ID NO:3 can be a componentof any fusion protein disclosed herein. In some embodiments, anextracellular portion of a PDGFR comprises the amino acid sequenceselected from the group consisting of SEQ ID NOs:7 and 8.

Amino acid sequence variants of any extracellular portion of a PDGFRprovided herein are also contemplated. For example, binding affinityand/or other biological properties of the extracellular portion of aPDGFR can be improved by altering the amino acid sequence encoding theprotein. Amino acids sequence variants of an extracellular portion of aPDGFR can be prepared by introducing appropriate modifications into thenucleic acid sequence encoding the protein or by introducing themodification by peptide synthesis. Such modifications include, forexample, deletions from, insertions into, and/or substitutions withinthe amino acid sequence of the extracellular portion of a PDGFR. Anycombination of deletion, insertion, and substitution can be made toarrive at the final amino acid construct of the extracellular portion ofa PDGFR provided that the final construct possesses the desiredcharacteristics such as binding to a PDGF family protein and/orinhibiting activation of the PDGF pathway. Accordingly, provided hereinare variants of an extracellular portion of a PDGFR that can be acomponent of any fusion protein disclosed herein. In some embodiments,an extracellular portion of a PDGFR comprises an amino acid sequencewith at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to the amino acid sequence of any one of Ig-likedomains D1, D2, D3, D4, or D5 of a PDGFR-α(e.g., human PDGFR-α). In someembodiments, an extracellular portion of a PDGFR comprises an amino acidsequence with at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to the amino acid sequence of any one ofIg-like domains D1, D2, D3, D4, or D5 of a PDGFR-β (e.g., humanPDGFR-β). In some embodiments, an extracellular portion of a PDGFRcomprises an amino acid sequence with at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to an aminoacid sequence selected from the group consisting of SEQ ID NOs:1-3. Insome embodiments, an extracellular portion of a PDGFR comprises an aminoacid sequence with at least 85%, at least 86%, at least 87%, at least88%, at least 89%, at least 90%, at least 91%, at least 92%, at least93%, at least 94%, at least 95%, at least 96%, at least 97%, at least98%, or at least 99% sequence identity to an amino acid sequenceselected from the group consisting of SEQ ID NOs:7 and 8.

Without being bound by theory, it is contemplated herein that anextracellular portion of a PDGFR inhibits activation of the PDGF pathwayby binding to a PDGF family protein to block its interaction with aPDGFR. Without being bound by theory, it is also contemplated hereinthat an extracellular portion of a PDGFR can bind to a PDGFR fordominant negative inhibition of the PDGF signaling pathway. In someaspects, an extracellular portion of a PDGFR binds a PDGF family proteinselected from the group consisting of PDGF-A, PDGF-B, PDGF-C, andPDGF-D. In some aspects, an extracellular portion of a PDGFR binds aPDGF family protein dimer selected from the group consisting of PPDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, and PDGF-DD. In some aspects, anextracellular portion of a PDGFR binds a PDGFR selected from the groupconsisting of PDGFR-α and PDGFR-β.

An extracellular portion of a PDGFR may or may not comprise a signalpeptide that serves as a signal sequence for secretion of theextracellular portion of a PDGFR from a host cell. The signal peptidecan be operably linked to a nucleic acid encoding the protein ofinterest (e.g., an extracellular portion of a PDGFR). In someembodiments, an extracellular portion of a PDGFR comprises a signalpeptide. In some embodiments, an extracellular portion of a PDGFR doesnot comprise a signal peptide.

Vascular Endothelial Growth Factor (VEGF) Receptor

There are at least five members of the VEGF family of proteins thatregulate the VEGF signaling pathway: VEGF-A, VEGF-B, VEGF-C, VEGF-D, andplacental growth factor (PlGF). Furthermore, alternative splicing ofmRNA that encodes VEGF-A, VEGF-B, and PlGF results in the generation ofmultiple isoforms of these proteins. For example, alternative splicingof VEGF-A yields nine different isoforms including isoforms VEGF₁₂₁,VEGF₁₆₅, VEGF₁₈₉, and VEGF₂₀₆. The VEGF family of proteins activate theVEGF signaling pathway by binding to the extracellular region oftransmembrane VEGF receptors. There are at least three identified VEGFreceptors: VEGFR1 (also known as fms-related tyrosine kinase 1 (Flt-1)),VEGFR2 (also known as kinase insert domain receptor (KDR)) and VEGFR3(also known as fms-like tyrosine kinase 4 (Flt-4)). VEGFRs each containan extracellular region comprising seven immunoglobulin (Ig)-likedomains, a single transmembrane domain segment, a juxtamembrane segment,and an intracellular protein-tyrosine kinase domain. The extracellularregions of VEGFRs bind to different members of the VEGF family ofproteins. For example, VEGFR1 binds VEGF-A, VEGF-B, and PlGF; VEGFR2binds all VEGF-A isoforms, VEGF-C, VEGF-D, and VEGF-E; and VEGFR3 bindsto VEGF-C and VEGF-D. See Roskoski, R et al., Crit. Rev Oncol Hematol.,2007, 62(3):179-213, which is incorporated herein by reference itsentirety, for a review of VEGF and VEGFR mediated signaling.

The present invention provides an extracellular portion of a VEGFreceptor that can be a component of any fusion protein disclosed herein.Accordingly, in one aspect, the invention provides for an extracellularportion of a VEGFR that includes, but is not limited to, VEGFR1, VEGFR2,and VEGFR3. In some of the embodiments herein, the VEGFR is from amammal, such as a human. There are seven extracellular Ig-like domainsnumbered 1, 2, 3, 4, 5, 6, and 7 starting from the N-terminus to theC-terminus of a VEGFR extracellular region. As used herein the terms“extracellular portion of a VEGFR” refers to one or more of the sevenIg-like domains in the VEGFR extracellular region. For example, “anextracellular portion of a VEGFR” refers to one or more of any of theseven Ig-like domains found in the extracellular region of a VEGFR suchas Ig-like domain D1, Ig-like domain D2, Ig-like domain D3, Ig-likedomain D4, Ig-like domain D5, Ig-like domain D6, or Ig-like domain D7.As used herein, terms such as “Ig-like domain D1” or “extracellulardomain (ECD) 1” of a VEGFR both specifically refer to the first Ig-likedomain found at the N-terminus of the extracellular region of VEGFR,“Ig-like domain D2” or “ECD 2” of a VEGFR both specifically refer to thesecond Ig-like domain from the N-terminus of the extracellular region ofVEGFR, and so forth. In any of the aspects herein, an extracellularportion of a VEGFR comprises at least one Ig-like domain of one or moreVEGFRs selected from the group consisting of VEGFR1, VEGFR2, and VEGFR3.In some aspects, an extracellular portion of a VEGFR comprises at least1, 2, 3, 4, 5, 6, but no more than 7 Ig-like domains of a VEGFR (e.g.,VEGFR1). In some aspects, an extracellular portion of a VEGFR comprises1 to 7, 1 to 6, 1 to 5, 1 to 4, 1 to 3, or 1 to 2 Ig-like domains of aVEGFR (e.g., VEGFR1). For example, an extracellular portion of a VEGFRcan comprise an Ig-like domain D2 of a VEGFR1. In another example, anextracellular portion of a VEGFR can comprise of Ig-like domains D1 toD3 of a VEGR1. In yet another example, an extracellular portion of aVEGFR can comprise the Ig-like domains D2 to D3 of VEGFR1 or the Ig-likedomains D1 to D3 of VEGFR2.

An extracellular portion comprising any combination of the seven Ig-likedomains of each VEGFR are contemplated herein. Accordingly, in oneaspect, the present invention provides an extracellular portion of aVEGFR comprising at least one Ig-like domain of two or more VEGFRs. Insome embodiments, an extracellular portion of a VEGFR comprises at leastone Ig-like domain from two or more VEGFRs selected from the groupconsisting of VEGFR1, VEGFR2, and VEGFR3. For example, a fusion proteinas described herein can comprise an extracellular portion of a VEGFRcomprising at least one Ig-like domain of VEGFR1 and at least oneIg-like domain of VEGFR2. In another example, a fusion protein asdescribed herein can comprise an extracellular portion of a VEGFRcomprising the Ig-like domain D2 of VEGFR1 and the Ig-like domains D3 toD4 of VEGFR2. In another example, a fusion protein as described hereincan comprise an extracellular portion of a VEGFR comprising the Ig-likedomain D2 of VEGFR1 and the Ig-like domain D3 of VEGFR3. In someaspects, an extracellular portion of a VEGFR comprises at least 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, but nomore than 21 Ig-like domains of at least two or more VEGFRs. In afurther aspect, an extracellular portion of a VEGFR comprises 1 to 21, 1to 20, 1 to 19, 1 to 18, 1 to 17, 1 to 16, 1 to 15, 1 to 14, 1 to 13, 1to 12, 1 to 11, 1 to 10, 1 to 9, 1 to 8, 1 to 7, 1 to 6, 1 to 5, 1 to 4,1 to 3, or 1 to 2 Ig-like domains of at least two or more VEGFRs. For afurther description of Ig-like domains that can be used as part of anextracellular portion of a VEGFR, see U.S. Pat. No. 7,928,072,WO2006113277, Davis-Smyth, T., et al., J Biol Chem, 1998, 273:3216-3222,Holash, J., et al., PNAS, 2002, 99(17):11393-11398, and Pechan, P., etal., Gene Ther, 2009, 16:10-16, all of which are incorporated in theirentirety by reference.

In some aspects, an extracellular portion of a VEGFR comprises the aminoacid sequence of SEQ ID NO:4. In some aspects, an extracellular portionof a VEGFR comprises the amino acid sequence of SEQ ID NO:5. Forexample, an extracellular portion of a VEGFR comprising the amino acidsequence of SEQ ID NO:4 or SEQ ID NO:5 can be a component of any fusionprotein disclosed herein.

Amino acid sequence variants of any extracellular portion of a VEGFRprovided herein are also contemplated. For example, binding affinityand/or other biological properties of the extracellular portion of aVEGFR can be improved by altering the amino acid sequence encoding theprotein. Amino acids sequence variants of an extracellular portion of aVEGFR can be prepared by introducing appropriate modifications into thenucleic acid sequence encoding the protein or by introducing themodification by peptide synthesis. Such modifications include, forexample, deletions from, insertions into, and/or substitutions withinthe amino acid sequence of the extracellular portion of a VEGFR. Anycombination of deletion, insertion, and substitution can be made toarrive at the final amino acid construct of the extracellular portion ofa VEGFR provided that the final construct possesses the desiredcharacteristics such as binding to a VEGF family protein and/orinhibiting activation of the VEGF pathway. Accordingly, provided hereinare variants of an extracellular portion of a VEGFR that can be acomponent of any fusion protein disclosed herein. In some embodiments,an extracellular portion of a VEGFR comprises an amino acid sequencewith at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least99% sequence identity to the amino acid sequence of any one of Ig-likedomains D1, D2, D3, D4, D5, D6, or D7 of a VEGFR1 (e.g., human VEGFR1).In some embodiments, an extracellular portion of a VEGFR comprises anamino acid sequence with at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceof any one of Ig-like domains D1, D2, D3, D4, D5, D6, or D7 of a VEGFR2(e.g., human VEGFR2). In some embodiments, an extracellular portion of aVEGFR comprises an amino acid sequence with at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence of any one of Ig-like domains D1, D2, D3, D4, D5, D6, orD7 of a VEGFR3 (e.g., human VEGFR3). In some embodiments, anextracellular portion of a VEGFR comprises an amino acid sequence withat least 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to an amino acid sequence selected from the groupconsisting of SEQ ID NOs:4 and 5.

Without being bound by theory, it is contemplated herein that anextracellular portion of a VEGFR inhibits activation of the VEGF pathwayby binding to a VEGF family protein to block its interaction with aVEGFR. Without being bound by theory, it is also contemplated hereinthat an extracellular portion of a VEGFR can bind to a VEGFR fordominant negative inhibition of the VEGF signaling pathway. In someaspects, an extracellular portion of a VEGFR binds a VEGF family proteinselected from the group consisting of VEGF-A, VEGF-B, VEGF-C, VEGF-D,and PlGF. In some aspects, an extracellular portion of a VEGFR binds aVEGFR (e.g., VEGFR1, VEGFR2, and/or VEGFR3).

An extracellular portion of a VEGFR may or may not comprise a signalpeptide that serves as a signal sequence for secretion of theextracellular portion of a VEGFR from a host cell. The signal peptidecan be operably linked to a nucleic acid encoding the protein ofinterest (e.g., an extracellular portion of a VEGFR). In someembodiments, an extracellular portion of a VEGFR comprises a signalpeptide. In some embodiments, an extracellular portion of a VEGFR doesnot comprise a signal peptide.

Multimerization Domain

The present invention provides a multimerization domain (e.g., an Fcregion of an antibody) that can be a component of any fusion proteindisclosed herein. Multimerization domains are those portions ofmultimeric proteins that promote the association of subunits to form,for example dimers, trimers, tetramers, and so forth. As used herein theterm “multimerizing domain” may be used to refer to a dimerizing domain,a trimerizing domain, a tetramerizing domain, and so forth. Fusionproteins comprising a multimerization domain can interact with otherfusion proteins comprising a multimerization domain to produce fusionprotein multimers (e.g., fusion protein dimers). For example, an IgG Fcregion is a dimerizing domain that can be fused to an extracellularportion of a PDGFR or an extracellular portion of VEGFR as disclosedherein. A fusion protein comprising an extracellular portion of a PDGFRand an IgG Fc region can dimerize with another fusion protein comprisingan IgG Fc region to produce a fusion protein dimer with multispecificityto at least a PDGF. A multimerization domain can be any polypeptide thatforms a multimer with another polypeptide. Multimerization domains thatcan be used are known in the art. See. U.S. Pat. No. 7,928,072 andWO2006/113277. For example, an Fc region of an IgG1 or IgG2 lambda heavychain, such as the CH3 domain alone or both the CH2 and CH3 domains, canbe used as a multimerization domain. Other Fc regions fromimmunoglobulin isotypes, such as IgA, IgM, IgD, or IgE can also be usedas multimerization domains. As used herein the term “Fc region” is usedto define a C-terminal region of an immunoglobulin heavy chain thatcontains at least a portion of the constant region. The term includesnative sequence Fc regions and variant Fc regions. In one embodiment, ahuman IgG heavy chain Fc region extends from Cys226, or from Pro230, tothe carboxyl-terminus of the heavy chain. However, the C-terminal lysine(Lys447) of the Fc region may or may not be present. In one embodiment,the multimerization domain is an Fc region of an antibody. In a furtherembodiment, the Fc region of an antibody is selected from the groupconsisting of an IgG Fc region, an IgA Fc region, an IgM Fc region, anIgD Fc region, and an IgE Fc region. In another further embodiment, theFc region of an antibody is selected from the group consisting of anIgG1 Fc region, an IgG2 Fc region, an IgG3 Fc region, and an IgG4 Fcregion. In some aspects, the Fc region comprises a CH3 region of IgG1,IgG2, IgG3, or IgG4. In some aspects, the Fc region comprises a CH2 anda CH3 region of IgG1, IgG2, IgG3, or IgG4. Amino acid sequences encodingimmunoglobulins that comprise Fc regions are well known in the art. Forexample, the IgG1 lambda heavy chain amino acid sequence can be foundunder Genbank accession no. CAA75032. An Fc region of an immunoglobulincan be obtained by cleavage with the enzyme papain or by other means. Insome embodiments, the Fc region comprises the amino acid sequence of SEQID NO:6. The multimerization domain of a VEGF can also be used such asthe multimerization domain of VEGF-A. VEGF-A is encoded by a nucleicacid shown at Genbank accession no. NM003376. For example, themultimerization domain of VEGF-A is encoded by VEGF-A exon 3 and can belinked to any of the fusion protein components disclosed herein such asthe extracellular portion of a PDGFR and/or the extracellular portion ofa VEGFR.

In some embodiments, amino acid sequence variants of a multimerizationdomain are provided herein. For example, it may be desirable to improvethe biological properties (e.g., multimerization properties) of themultimerization domain. Amino acids sequence variants of amultimerization domain can be prepared by introducing appropriatemodifications into the nucleic acid sequence encoding the protein or byintroducing the modification by peptide synthesis. Such modificationsinclude, for example, deletions from, insertions into, and/orsubstitutions within the amino acid sequence of the multimerizationdomain. Any combination of deletion, insertion, and substitution can bemade to arrive at the final amino acid construct of the multimerizationprovided that the final construct possesses the desired characteristicssuch as formation of multimer proteins. Accordingly, provided herein arevariants of a multimerization domain (e.g., an Fc region of an antibody)that can be a component of any fusion protein disclosed herein. In someembodiments, an Fc region comprises an amino acid sequence with at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence of a CH3 region of IgG1, IgG2, IgG3,or IgG4. In some embodiments, an Fc region comprises an amino acidsequence with at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, or atleast 99% sequence identity to the amino acid sequence of a CH2 and aCH3 region of IgG1, IgG2, IgG3, or IgG4. In some embodiments, an Fcregion comprises an amino acid sequence with at least 85%, at least 86%,at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence of SEQ ID NO:6. Variants of multimerization domains arewell known in the art. See for example U.S. Patent Application No.2012/0251531, which is incorporated herein by reference in its entirety.

Linkers

Components of the fusion protein (e.g., the extracellular portion of aPDGFR, the extracellular portion of a VEGFR, or the multimerizationdomain) may be linked by a linking moiety such as a peptide linker.Preferably, the linker increases flexibility of the fusion proteincomponents and does not interfere significantly with the structure ofeach functional component within the fusion protein. In someembodiments, the linker moiety is a peptide linker. In some embodiments,the peptide linker comprises 2 to 100 amino acids. In some embodiments,the peptide linker comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14,15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32,33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50,51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86,87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99 but no greater than100 amino acids. In some embodiments, the peptide linker is between 5 to75, 5 to 50, 5 to 25, 5 to 20, 5 to 15, 5 to 10 or 5 to 9 amino acids inlength. Exemplary linkers include linear peptides having at least twoamino acid residues such as Gly-Gly, Gly-Ala-Gly, Gly-Pro-Ala,Gly-Gly-Gly-Gly-Ser. Suitable linear peptides include poly glycine,polyserine, polyproline, polyalanine and oligopeptides consisting ofalanyl and/or serinyl and/or prolinyl and/or glycyl amino acid residues.In some embodiments, the peptide linker comprises the amino acidsequence selected from the group consisting of Gly₉, Glu₉, Ser₉,Gly₅-Cys-Pro₂-Cys, (Gly₄-Ser)₃,Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn,Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn,Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys, andGly₉-Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn.

Linker moieties can also be made from other polymers, such aspolyethylene glycol. Such linkers can have from 10 to 1000, 10 to 500,10 to 250, 10 to 100, or 10 to 50 ethylene glycol monomer units.Suitable polymers should be of a size similar to the size occupied bythe appropriate range of amino acid residues. A typical sized polymerwould provide a spacing of from about 10-25 angstroms.

The linker moiety may be a protein multivalent linker that has branched“arms” that link multiple fusion protein components in a non-linearfashion. In some embodiments, a multivalent linker has about 3 to 40amino acid residues, all or some of which provide attachment sites forconjugation with fusion protein components (e.g., the extracellularportion of a PDGFR, the extracellular portion of a VEGFR, or themultimerization domain). Alpha amino groups and alpha carboxylic acidscan serve as attachment sites. Exemplary multivalent linkers include,but are not limited to, polylysines, polyornithines, polycysteines,polyglutamic acid and polyaspartic acid. Optionally, amino acid residueswith inert side chains, e.g., glycine, alanine and valine, can beincluded in the amino acid sequence. The linkers may also be anon-peptide chemical entity such as a chemical linker that is suitablefor administration (e.g., ocular administration) once attached to afusion protein component (e.g., the extracellular portion of a PDGFR,the extracellular portion of a VEGFR, and/or the multimerizationdomain). The chemical linker may be a bifunctional linker, each of whichreacts with a fusion protein component (e.g., the extracellular portionof a PDGFR, the extracellular portion of a VEGFR, and/or themultimerization domain). Alternatively, the chemical linker may be abranched linker that has a multiplicity of appropriately spaced reactivegroups, each of which can react with a functional group of a fusionprotein component (e.g., the extracellular portion of a PDGFR, theextracellular portion of a VEGFR, and/or the multimerization domain).The fusion protein components (e.g., the extracellular portion of aPDGFR, the extracellular portion of a VEGFR, and/or the multimerizationdomain) are attached by way of reactive functional groups and are spacedsuch that steric hindrance does not substantially interfere withformation of covalent bonds between some of the reactive functionalgroups (e.g., amines, carboxylic acids, alcohols, aldehydes and thiols)and the peptide. Examples of linker moieties include, but are notlimited to, those disclosed in Tarn, J. P., et al., J. of ImmunolMethods, 1996, 196:17-32.

The linker moieties may be used to link any of the components of thefusion proteins disclosed herein. For example, a peptide linker (e.g.,Gly₉) can be used to link the C-terminus end of an extracellular portionof a PDGFR to the N-terminus end of an extracellular portion of a VEGFRand can be further used to link the C-terminus end of the extracellularportion of a VEGR to the N-terminus end of a multimerization domain(e.g., an IgG1 Fc region). In some embodiments, a linker is used betweenan extracellular portion of a PDGFR and a multimerization domain. Insome embodiments, a linker is used between an extracellular portion of aVEGFR and a multimerization domain. In some embodiments, a linker isused between an extracellular portion of a PDGFR and an extracellularportion of a VEGFR. In some embodiments, the fusion protein comprises alinker between an extracellular portion of a PDGFR and an extracellularregion of a VEGFR, and a linker between the extracellular region of theVEGFR and a multimerization domain (e.g., Fc region). In someembodiments, a fusion protein comprises at least one linker but no morethan four linkers. For example, a fusion protein can comprise (a) anextracellular portion of a PDGFR, (b) an extracellular portion of aVEGFR, (c) a multimerization domain (e.g., an IgG1 Fc region), and atleast one linker from the N-terminus to the C-terminus in an orderselected from the group consisting of: (1) linker, a, linker, b, linker,c, linker; (2) a, linker, b, linker, c, linker; (3) linker, a, linker,b, linker, c; (4) a, linker, b, linker, c; (5) a, linker, b, c; and (6)a, b, linker, c. In another example, a fusion protein can comprise (a)an extracellular portion of a PDGFR, (b) a multimerization domain (e.g.,an IgG1 Fc region), and at least one linker from the N-terminus to theC-terminus in an order selected from the group consisting of: (1)linker, a, linker, b, linker; (2) linker, a, linker, b; (3) a, b,linker; (4) a, linker, b; (5) linker, b, linker, a, linker; (6) linker,b, linker, a; (7) b, a, linker; and (8) b, linker, a.

Fusion Proteins

Provided herein are fusion proteins that have binding specificities toat least two different binding partners (e.g., PDGF and VEGF). In someembodiments, a fusion protein comprises a first binding specificity to aprotein of the PDGF family (e.g., PDGF-A, PDGF-B, PDGF-C, or PDGF-D) anda second binding specificity to a VEGF (e.g., VEGF-A VEGF-B, VEGF-C,VEGF-D, or PlGF). In some embodiments, a fusion protein comprises afirst binding specificity to a protein dimer of the PDGF family (e.g.,PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, or PDGF-DD) and a second bindingspecificity to a VEGF (e.g., VEGF-A VEGF-B, VEGF-C, VEGF-D, or PlGF). Insome embodiments, a fusion protein comprises a first binding specificityto a mammalian (e.g., human) PDGF and a second binding specificity to amammalian (e.g., human) VEGF. In some embodiments, a fusion proteinbinds to the same PDGF as any of the PDGFRs described herein. In someembodiments, a fusion protein binds to the same component of the PDGFpathway as any one of PDGFR-α or PDGFR-β. In some embodiments, a fusionprotein binds to the same PDGF as any one of PDGFR-α/PDGFR-α,PDGFR-β/PDGFR-β, or PDGFR-α/PDGFR-β dimers. In some embodiments, afusion protein comprises at least one extracellular portion of a PDGFRof any of the PDGFRs described herein. For example, a fusion protein cancomprise at least one extracellular portion of PDGFR-α and at least oneextracellular portion of PDGFR-β. In another example, a fusion proteincan comprise two extracellular portions of PDGFR-β such as Ig-likedomain D1-D3 and Ig-like domain D1-D5. In some aspects, a fusion proteincomprises an extracellular portion of a PDGFR comprising the amino acidsequence selected from the group consisting of SEQ ID NOs: 1-3. In someaspects, a fusion protein comprises an extracellular portion of a PDGFRcomprising the amino acid sequence selected from the group consisting ofSEQ ID NOs:7 and 8. In some embodiments, a fusion protein binds to thesame component of the VEGF pathway as any of the VEGFRs describedherein. In some embodiments, a fusion protein binds to the samecomponent of the VEGF pathway as any one of VEGFR1, VEGFR2, or VEGFR3.In some embodiments, a fusion protein comprises at least oneextracellular portion of a VEGFR of any of the VEGFRs described herein.For example, a fusion protein can comprise at least one extracellularportion of VEGFR1 and at least one extracellular portion of VEGFR2. Inanother example, a fusion protein can comprise two extracellularportions of VEGFR1 such as Ig-like domain D2 and Ig-like domain D1-D3.In some aspects, a fusion protein comprises an extracellular portion ofa VEGFR comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOs:4 and 5. Any of the fusion proteins disclosedherein comprising an extracellular portion of a PDGFR and anextracellular portion of a VEGFR can further comprise a multimerizationdomain. In some embodiments, the multimerization domain is an Fc region(e.g., an IgG1 Fc region). In some embodiments, the Fc region comprisesthe amino acid sequence of SEQ ID NO:6. In some embodiments, the fusionprotein comprising an extracellular portion of a PDGFR, an extracellularportion of VEGFR, and a multimerization domain inhibits the PDGF andVEGF signaling pathways (e.g., inhibition of PDGF and VEGF activity).Any of the fusion proteins disclosed herein comprising an extracellularportion of a PDGFR, an extracellular portion of VEGFR, and amultimerization domain can further comprise a linker. The linker can beany linker as disclosed herein. In some embodiments, the linker is apeptide linker. In some embodiments, the linker comprises the amino acidsequence selected from the group consisting of Gly₉, Glu₉, Ser₉,Gly₅-Cys-Pro₂-Cys, (Gly₄-Ser)₃,Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn,Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn,Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys, andGly₉-Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn. In someembodiments, the extracellular portion of a PDGFR comprises anextracellular portion of a mammalian (e.g., human) PDGFR. In someembodiments, the extracellular portion of a VEGFR comprises anextracellular portion of a mammalian (e.g., human) VEGFR. In someembodiments, a fusion protein comprises an extracellular portion of ahuman PDGFR (e.g., human PDGFR-β) and an extracellular portion of ahuman VEGFR (e.g., human VEGFR1).

In one aspect, the invention provides a fusion protein comprising: a) anextracellular portion of a PDGFR comprising the amino acid sequence ofSEQ ID NO:1, 2, 3, 7, or 8; b) an extracellular portion of a VEGFRcomprising the amino acid sequence of SEQ ID NO:4 or 5; and c) amultimerization domain comprising the amino acid sequence of SEQ IDNO:6. In some embodiments, the fusion protein comprises: a) anextracellular portion of a PDGFR comprising the amino acid sequence ofSEQ ID NO:1; b) an extracellular portion of a VEGFR comprising the aminoacid sequence of SEQ ID NO:4; and c) a multimerization domain comprisingthe amino acid sequence of SEQ ID NO:6. In some embodiments, the fusionprotein comprises: a) an extracellular portion of a PDGFR comprising theamino acid sequence of SEQ ID NO:3; b) an extracellular portion of aVEGFR comprising the amino acid sequence of SEQ ID NO:4; and c) amultimerization domain comprising the amino acid sequence of SEQ IDNO:6.

Provided herein are fusion proteins comprising an extracellular portionof a PDGFR, an extracellular portion of a VEGFR, and a multimerizationdomain in a specific order. In some embodiments, the fusion proteincomprises (a) an extracellular portion of a PDGFR, (b) an extracellularportion of a VEGFR, and (c) a Fc region arranged from the N-terminus toC-terminus in an order of a, b, c. In some of the embodiments, anextracellular portion of a PDGFR comprises the Ig-like domains D1-D3 ofa PDGFR (e.g., PDGFR-β). In some embodiments, an extracellular portionof a PDGFR comprises the Ig-like domains D 1-D4 of a PDGFR (e.g.,PDGFR-β). In some embodiments, an extracellular portion of a PDGFRcomprises the Ig-like domains D1-D5 of a PDGFR (e.g., PDGFR-β). In someembodiments, an extracellular portion of a VEGFR comprises the Ig-likedomain D2 of a VEGFR (e.g., VEGFR1). In some embodiments, anextracellular portion of a VEGFR comprises the Ig-like domains D1-D3 ofa VEGFR (e.g., VEGFR1). In some embodiments, a multimerization domaincomprises the Fc region of an IgG1 antibody.

In some embodiments, the fusion protein comprises the amino acidsequence of SEQ ID NO:12. In other embodiments, the fusion proteincomprises the amino acid sequence of SEQ ID NO:13. In still otherembodiments, the fusion protein comprises the amino acid sequence of SEQID NO:14. In yet other embodiments, the fusion protein comprises theamino acid sequence of SEQ ID NO:15.

Fusion proteins comprising at least two or more extracellular portionsof a PDGFR, two or more extracellular portions of a VEGFR, and/or two ormore multimerization domains are also contemplated. For example, afusion protein may comprise (a) an extracellular portion of a PDGFR, (b)an extracellular portion of a VEGFR, and (c) a Fc region arranged fromthe N-terminus to C-terminus in an order of a, a, b, c or in an order ofa, b, b, c. Any combination of at least one extracellular portion of aPDGFR, at least one extracellular portion of a VEGFR, and at least onemultimerization domain is provided herein as if each combination hadbeen expressly stated herein.

Fusion proteins comprising an extracellular portion of a PDGFR and amultimerization domain are also contemplated. In some embodiments, thefusion protein comprises (a) an extracellular portion of a PDGFR and (b)a Fc region arranged from the N-terminus to C-terminus in an order of aand b. In some embodiments, the fusion protein comprises (a) anextracellular portion of a PDGFR and (b) a Fc region arranged from theN-terminus to C-terminus in an order of b and a. In some embodiments, anextracellular portion of a PDGFR comprises the Ig-like domains D1-D2 ofa PDGFR (e.g., PDGFR-β). In some embodiments, an extracellular portionof a PDGFR comprises the Ig-like domains D1-D3 of a PDGFR (e.g.,PDGFR-β). In some embodiments, an extracellular portion of a PDGFRcomprises the Ig-like domains D1-D4 of a PDGFR (e.g., PDGFR-β). In someembodiments, an extracellular portion of a PDGFR comprises the Ig-likedomains D1-D5 of a PDGFR (e.g., PDGFR-β). In some embodiments, amultimerization domain comprises the Fc region of an IgG1 antibody. Anycombination of at least one extracellular portion of a PDGFR and atleast one multimerization domain is provided herein as if eachcombination had been expressly stated herein.

In some embodiments, the fusion protein comprises the amino acidsequence of SEQ ID NO:9. In some embodiments, the fusion proteincomprises the amino acid sequence of SEQ ID NO:10. In some embodiments,the fusion protein comprises the amino acid sequence of SEQ ID NO:11.

The fusion proteins described in the present invention can comprisemodified forms of the extracellular portion of a PDGFR, theextracellular portion of a VEGFR, and/or the multimerization domain. Forexample, the fusion protein components can have post-translationalmodifications, including for example, glycosylation, sialylation,acetylation, and phosphorylation.

In some embodiments, amino acid sequence variants of the fusion proteinsare provided herein. For example, it may be desirable to improve thebinding affinity and/or other biological properties of the extracellularportion of a PDGFR, the extracellular portion of a VEGFR, and/or themultimerization domain. Amino acid sequence variants of the fusionprotein may be prepared by introducing appropriate modifications intothe nucleotide sequence encoding the extracellular portion of a PDGFR,the extracellular portion of a VEGFR, and/or the multimerization domain,or by introduction through peptide synthesis. Such modificationsinclude, for example, deletions from, insertions into, and/orsubstitutions of residues within the amino acid sequences of theextracellular portion of a PDGFR, the extracellular portion of a VEGFR,and/or the multimerization domain. Any combination of deletion,insertion, and substitution can be made to arrive at the finalconstruct, provided that the final construct possesses the desiredcharacteristics (e.g., binding to a PDGF, binding to a VEGF, inhibitingactivation of a PDGF pathway, multimer formation, and/or inhibitingactivation of a VEGF pathway). In some embodiments, the fusion proteincomprises at least 85%, at least 86%, at least 87%, at least 88%, atleast 89%, at least 90%, at least 91%, at least 92%, at least 93%, atleast 94%, at least 95%, at least 96%, at least 97%, at least 98%, atleast 99% sequence identity to the amino acid sequence of a fusionprotein comprising any extracellular portion of a PDGFR, extracellularportion of a VEGFR, and/or multimerization domain as disclosed herein.In some embodiments, a fusion protein variant comprises at least 85%, atleast 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto the amino acid sequence selected from the group consisting of SEQ IDNOs:12-15. In some embodiments, a fusion protein variant comprises atleast 85%, at least 86%, at least 87%, at least 88%, at least 89%, atleast 90%, at least 91%, at least 92%, at least 93%, at least 94%, atleast 95%, at least 96%, at least 97%, at least 98%, or at least 99%sequence identity to the amino acid sequence selected from the groupconsisting of SEQ ID NOs:9-11.

Amino acid residue substitutions disclosed herein also includeconservative substitutions. Conservative substitutions are shown in theTable 1 below under the heading of “conservative substitutions”. If suchsubstitutions result in a change in biological activity, then moresubstantial changes, denominated “exemplary substitutions” in Table 1,or as further described below in reference to amino acid classes, may beintroduced and the products screened. Amino acid substitutions as shownin Table 1 or as described below in reference to the amino acid classesmay be introduced into any of the fusion proteins or protein components(e.g., extracellular portion of a PDGFR, extracellular portion of aVEGFR, multimerization domain, etc.) provided herein.

TABLE 1 Potential amino acid substitutions Original ExemplaryConservative Residue Substitutions Substitutions Ala (A) Val; Leu; IleVal Arg (R) Lys; Gln; Asn Lys Asn (N) Gln; His; Asp, Lys; Arg Gln Asp(D) Glu; Asn Glu Cys (C) Ser; Ala Ser Gln (Q) Asn; Glu Asn Glu (E) Asp;Gln Asp Gly (G) Ala Ala His (H) Asn; Gln; Lys; Arg Arg Ile (I) Leu; Val;Met; Ala; Phe; Norleucine Leu Leu (L) Norleucine; Ile; Val; Met; Ala;Phe Ile Lys (K) Arg; Gln; Asn Arg Met (M) Leu; Phe; Ile Leu Phe (F) Trp;Leu; Val; Ile; Ala; Tyr Tyr Pro (P) Ala Ala Ser (S) Thr Thr Thr (T) Val;Ser Ser Trp (W) Tyr; Phe Tyr Tyr (Y) Trp; Phe; Thr; Ser Phe Val (V) Ile;Leu; Met; Phe; Ala; Norleucine Leu

Substantial modifications in the biological properties of the proteinsor polypeptides are accomplished by selecting substitutions that differsignificantly in their effect on maintaining (a) the structure of thepolypeptide backbone in the area of the substitution, for example, as asheet or helical conformation, (b) the charge or hydrophobicity of themolecule at the target site, or (c) the bulk of the side chain. Aminoacids may be grouped according to common side-chain properties:

-   -   (1) hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile;    -   (2) neutral hydrophilic: Cys, Ser, Thr, Asn, Gln;    -   (3) acidic: Asp, Glu;    -   (4) basic: His, Lys, Arg;    -   (5) residues that influence chain orientation: Gly, Pro;    -   (6) aromatic: Trp, Tyr, Phe;    -   (7) large hydrophobic: Norleucine, Met, Val, Leu, Ile.

Non-conservative substitutions entail exchanging a member of one ofthese classes for another class.

A useful method for identification of certain residues or regions of thefusion protein that are preferred locations for mutagenesis is called“alanine scanning mutagenesis” as described by Cunningham and Wells inScience, 1989, 244:1081-1085. Here, a residue or group of targetresidues are identified (e.g., charged residues such as arg, asp, his,lys, and glu) and replaced by a neutral or negatively charged amino acid(most preferably alanine or polyalanine) to affect the interaction ofthe amino acids with the target binding partner. Those amino acidlocations demonstrating functional sensitivity to the substitutions thenare refined by introducing further or other variants at, or for, thesites of substitution. Thus, while the site for introducing an aminoacid sequence variation is predetermined, the nature of the mutation perse need not be predetermined. For example, to analyze the performance ofa mutation at a given site, ala scanning or random mutagenesis isconducted at the target codon or region and the expressed fusionpolypeptide variants are screened for the desired activity.

Any cysteine residue not involved in maintaining the proper conformationof the fusion proteins or protein components (e.g., extracellularportion of a PDGFR, extracellular portion of a VEGFR, multimerizationdomain, etc.) also may be substituted, generally with serine, to improvethe oxidative stability of the molecule and prevent aberrantcrosslinking. Conversely, cysteine bond(s) may be added to the fusionprotein or protein components (e.g., extracellular portion of a PDGFR,extracellular portion of a VEGFR, multimerization domain, etc.) toimprove its stability.

In further embodiments, proteins or peptides of the invention maycomprise one or more non-naturally occurring or modified amino acids. A“non-naturally occurring amino acid residue” refers to a residue, otherthan those naturally occurring amino acid residues listed above, whichis able to covalently bind adjacent amino acid residues(s) in apolypeptide chain. Non-natural amino acids include, but are not limitedto homo-lysine, homo-arginine, homo-serine, azetidinecarboxylic acid,2-aminoadipic acid, 3-aminoadipic acid, beta-alanine, aminopropionicacid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid,2-aminoheptanoic acid, 2-aminoisobutyric acid, 3-aminoisbutyric acid,2-aminopimelic acid, tertiary-butylglycine, 2,4-diaminoisobutyric acid,desmosine, 2,2′-diaminopimelic acid, 2,3-diaminopropionic acid,N-ethylglycine, N-ethylasparagine, homoproline, hydroxylysine,allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine,allo-isoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine,N-methylpentylglycine, N-methylvaline, naphthalanine, norvaline,norleucine, ornithine, citrulline, pentylglycine, pipecolic acid andthioproline. Modified amino acids include natural and non-natural aminoacids which are chemically blocked, reversibly or irreversibly, ormodified on their N-terminal amino group or their side chain groups, asfor example, N-methylated D and L amino acids, side chain functionalgroups that are chemically modified to another functional group. Forexample, modified amino acids include methionine sulfoxide; methioninesulfone; aspartic acid- (beta-methyl ester), a modified amino acid ofaspartic acid; N-ethylglycine, a modified amino acid of glycine; oralanine carboxamide and a modified amino acid of alanine. Additionalnon-natural and modified amino acids, and methods of incorporating theminto proteins and peptides, are known in the art (see, e.g., Sandberg etal., (1998) J. Med. Chem. 41: 2481-91; Xie and Schultz (2005) Curr.Opin. Chem. Biol. 9: 548-554; Hodgson and Sanderson (2004) Chem. Soc.Rev. 33: 422-430.

Amino acid sequence insertions include amino- (“N”) and/or carboxy-(“C”) terminal fusions ranging in length from one residue to a hundredor more residues, as well as intrasequence insertions of single ormultiple amino acid residues. Examples of terminal insertions include afusion protein with an N-terminal methionyl residue or the fusionprotein fused to a cytotoxic polypeptide. Other insertional variants ofthe fusion protein molecule include fusion to the N- or C-terminus ofthe fusion protein a polypeptide that allows formation of proteinmultimers.

The present invention provides a signal peptide, also referred herein asa signal sequence, which can be a component of any fusion proteinprovided herein. For example, a fusion protein comprising anextracellular portion of a PDGFR, an extracellular portion of a VEGFR,and a multimerization domain may further comprise a heterologouspeptide, preferably a signal sequence or other peptide having a specificcleavage site at the N-terminus of the mature fusion protein. Theheterologous signal sequence selected preferably is one that isrecognized and processed (i.e., cleaved by a signal peptidase) byeukaryotic host-cells. For prokaryotic host-cells that do not recognizeand process native mammalian signal sequences, the eukaryotic (i.e.,mammalian) signal sequence is replaced by a prokaryotic signal sequenceselected, for example, from the group consisting of leader sequencesfrom alkaline phosphatase, penicillinase, lpp, or heat-stableenterotoxin II genes. For yeast secretion the native signal sequence maybe substituted by, e.g., the yeast invertase leader, factor leader(including Saccharomyces and Kluyveromyces-factor leaders), or acidphosphatase leader, the C. albicans glucoamylase leader, or the signaldescribed in WO 90/13646. In mammalian cell expression, mammalian signalsequences as well as viral secretory leaders, for example, the herpessimplex virus gD signal, are available. A signal peptide can becompletely cleaved from the fusion protein as it is produced from hostcells or it can be partially cleaved. A mixed population of fusionproteins can be produced from a host cell wherein fusion proteinscomprise a completely cleaved signal sequence (e.g., no signalsequence), a partially cleaved signal sequence (e.g., portion of thesignal sequence) and/or a non-cleaved signal sequence (e.g., completesignal sequence). For example, a fusion protein further comprising asignal peptide at the N-terminus can be cleaved at the N-terminus by anyone of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18,19, 20, 21, or 22 amino acid residues. In some embodiments, any fusionprotein described herein comprises a signal peptide for proteinsecretion from a cell. In some embodiments, any fusion protein describedherein does not comprise a signal peptide for protein secretion from acell.

The present invention provides a dimeric fusion protein comprising twofusion proteins, wherein each fusion protein comprises any fusionprotein disclosed herein. In one embodiment, the dimeric fusion proteincomprises two identical fusion proteins. In another embodiment, thedimeric fusion protein comprises two different fusion proteins. Thefusion proteins disclosed herein may form multimers of two or morefusion proteins. Multimers (e.g., dimers, trimers, tetramers, etc.) canform from identical fusion proteins (e.g., homomultimer) or formheterologous fusion proteins (e.g., heteromultimer). In anotherembodiment, the multimeric fusion protein comprises at least one fusionprotein comprising the amino acid sequence selected from the groupconsisting of SEQ ID NOs:12-15, or an amino acid sequence having atleast 90% identity to the amino acid sequence selected from the groupconsisting of SEQ ID NOs:12-15. In another embodiment, the multimericfusion protein comprises at least one fusion protein comprising theamino acid sequence selected from the group consisting of SEQ IDNOs:9-11, or an amino acid sequence having at least 90% identity to theamino acid sequence selected from the group consisting of SEQ IDNOs:9-11. In an embodiment, the fusion protein is recovered as a proteinfusion multimer from a host cell comprising a nucleic acid encoding saidfusion protein. In some embodiments, the fusion proteins areglycosylated. For example, the fusion protein may be glycosylated afterrelease from a host cell at the extracellular portion of a PDGFR, theextracellular portion of a VEGFR, and/or multimerization domain.

Also provided herein are pharmaceutical compositions comprising a fusionprotein of the invention and a pharmaceutically acceptable carrier. Thepharmaceutical compositions may be suitable for a variety of modes ofadministration described herein, including for example systemic orlocalized administration. The pharmaceutical compositions can be in theform of eye drops, injectable solutions, or in a form suitable forinhalation (either through the mouth or the nose) or oraladministration. In some embodiments, the pharmaceutical compositionscomprising a fusion protein described herein and a pharmaceuticallyacceptable carrier is suitable for administration to human. In someembodiments, the pharmaceutical compositions comprising a fusion proteindescribed herein and a pharmaceutically acceptable carrier is suitablefor intravitreal injection or topical application to the eye. Suchpharmaceutically acceptable carriers can be sterile liquids, such aswater and oil, including those of petroleum, animal, vegetable orsynthetic origin, such as peanut oil, soybean oil, mineral oil, and thelike. Saline solutions and aqueous dextrose, polyethylene glycol (PEG)and glycerol solutions can also be employed as liquid carriers,particularly for injectable solutions. The pharmaceutical compositionmay further comprise additional ingredients, for example preservatives,buffers, tonicity agents, antioxidants and stabilizers, nonionic wettingor clarifying agents, viscosity-increasing agents, and the like. Thepharmaceutical compositions described herein can be packaged in singleunit dosages or in multidosage forms. The compositions are generallyformulated as sterile and substantially isotonic solution. Compositionscan also be formulated to have osmotic values that are compatible withthe aqueous humor of the eye and ophthalmic tissues. Such osmotic valueswill generally be in the range of from about 200 to about 400milliosmoles per kilogram of water (“mOsm/kg”), but will preferably beabout 300 mOsm/kg. The retina is considered to have an osmotic value of˜283 mOsm/kg.

IV. Nucleic Acids, Vectors, and Host Cells

Nucleic Acids

Provided herein are isolated nucleic acids encoding any of the fusionproteins components disclosed herein, such as an extracellular portionof a PDGFR, and extracellular portion of a VEGFR, and a multimerizationdomain. Nucleic acids encoding mammalian PDGFR have been described forboth receptor types, PDGFR-α and PDGFR-β. Exemplary nucleic acidsequences can be found at, but are not limited to, Yarden et al.,Nature, 1986, 323:226-232; Matsui et al., Science, 1989, 243: 800-803;U.S. patent application Ser. No. 07/771,829 which is a continuation ofU.S. patent application Ser. No. 07/309,332, now abandoned, U.S. Pat.No. 5,686,572, and WO2006/113277. mRNA encoding human PDGFR-α andPDGFR-β can be found at Genbank Accession Nos. NM_(—)006206.4 andNM_(—)002609.3, respectively. In some embodiments, an isolated nucleicacid encodes an extracellular portion of a PDGFR comprising an aminoacid sequence selected from the group consisting of SEQ ID NOs:1-3. Insome embodiments, an isolated nucleic acid encodes an extracellularportion of a PDGFR comprising an amino acid sequence with at least 85%,at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, atleast 91%, at least 92%, at least 93%, at least 94%, at least 95%, atleast 96%, at least 97%, at least 98%, or at least 99% sequence identityto the amino acid sequence selected from the group consisting of SEQ IDNOs:1-3. In some embodiments, the isolated nucleic acid encoding anextracellular portion of a PDGFR is selected from the group consistingof SEQ ID NOs:16 and 17. Also provided herein are isolated nucleic acidsencoding an extracellular portion of a VEGFR. Nucleic acids encodingmammalian VEGFR have been described for all receptor types. Exemplarynucleic acid sequences can be found at, but are not limited to, U.S.Pat. No. 7,928,072 and WO2006/113277. mRNA encoding human VEGFR1 andVEGFR2 can be found at Genbank Accession Nos. NM_(—)001159920.1 andNM_(—)002253.2, respectively. mRNA encoding human VEGFR3 can be found atGenbank Accession Nos. NM_(—)002020.7 and NM_(—)182925.4. In someembodiments, an isolated nucleic acid encodes an extracellular portionof a VEGFR comprising an amino acid sequence selected from the groupconsisting of SEQ ID NOs:4 and 5. In some embodiments, an isolatednucleic acid encodes an extracellular portion of a VEGFR comprising anamino acid sequence with at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, or at least 99% sequence identity to the amino acid sequenceselected from the group consisting of SEQ ID NOs:4 and 5. Also providedherein are isolated nucleic acids encoding a multimerization domain(e.g., Fc region). In some embodiments, an isolated nucleic acid encodesa multimerization domain comprising an amino acid sequence of SEQ IDNO:6. In some embodiments, an isolated nucleic acid encodes amultimerization domain comprising an amino acid sequence with at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, or at least 99% sequenceidentity to the amino acid sequence selected from the group consistingof SEQ ID NO:6.

Also provided are isolated nucleic acids encoding a fusion protein asdisclosed herein. In some embodiments, an isolated nucleic acid encodesa fusion protein comprising an amino acid sequence selected from thegroup consisting of SEQ ID NOs:12-15. In some embodiments, an isolatednucleic acid encodes a fusion protein comprising an amino acid sequenceselected from the group consisting of SEQ ID NOs:9-11. In someembodiments, an isolated nucleic acid encodes a fusion proteincomprising an amino acid sequence with at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence selected from the group consisting of SEQ ID NOs:12-15. Insome embodiments, an isolated nucleic acid encodes a fusion proteincomprising an amino acid sequence with at least 85%, at least 86%, atleast 87%, at least 88%, at least 89%, at least 90%, at least 91%, atleast 92%, at least 93%, at least 94%, at least 95%, at least 96%, atleast 97%, at least 98%, or at least 99% sequence identity to the aminoacid sequence selected from the group consisting of SEQ ID NOs:9-11. Insome embodiments, an isolated nucleic acid encoding a fusion proteincomprises the nucleic acid sequence selected from the group consistingof SEQ ID NOs:21-24. In some embodiments, an isolated nucleic acidencoding a fusion protein comprises the nucleic acid sequence selectedfrom the group consisting of SEQ ID NOs:18-20.

An isolated nucleic acid sequence encoding a fusion protein or acomponent of a fusion protein (e.g., the extracellular portion of aPDGFR, the extracellular portion of a VEGFR, or the multimerizationdomain) may further include a nucleic acid sequence encoding a linker.In some embodiments, a nucleic acid encodes a linker selected from thegroup consisting of Gly₉, Glu₉, Ser₉, Gly₅-Cys-Pro₂-Cys, (Gly₄-Ser)₃,Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn,Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn,Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys, andGly₉-Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn.

Isolated nucleic acids may further include a sequence encoding a signalpeptide that serves as a signal sequence to secrete the fusion proteinfrom the host cells. In some embodiments, the isolated nucleic acid doesnot comprise a sequence encoding a signal peptide.

Isolated nucleic acid molecules encoding a fusion protein or a componentof a fusion protein (e.g., the extracellular portion of a PDGFR, theextracellular portion of a VEGFR, or the multimerization domain) can bein the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or inthe form of DNA, including, but not limited to, cDNA and genomic DNAobtained by cloning or produced synthetically, or any combinationsthereof. The DNA can be triple-stranded, double-stranded orsingle-stranded, or any combination thereof. Any portion of at least onestrand of the DNA or RNA can be the coding strand, also known as thesense strand, or it can be the non-coding strand, also referred to asthe anti-sense strand. The isolated nucleic acids can be obtained frombiological sources using any number of cloning methodologies known tothose of skill in the art. The isolated nucleic acids can also beprepared by direct chemical synthesis by known methods. Nucleic acidsencoding a fusion protein or fusion protein component (e.g., theextracellular portion of a PDGFR, the extracellular portion of a VEGFR,or the multimerization domain) can be prepared by a variety of methodsknown in the art including, but not limited to, isolation from a naturalsource or preparation by oligonucleotide-mediated mutagenesis,site-directed mutagenesis, PCR mutagenesis, and cassette mutagenesis ofan earlier prepared variant or a non-variant version of the fusionprotein or fusion protein component. See Molecular Cloning: A LaboratoryManual (Sambrook et al., 4^(th) ed., Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y., 2012) and Current Protocols inMolecular Biology (F. M. Ausubel, et al. eds., 2003).

Vectors

The present invention contemplates the use of a nucleic acid deliveryvehicle for introduction of one or more nucleic acid sequences encodingfor a fusion protein or fusion protein component into a cell forexpression of said protein. Examples of nucleic acid delivery vehiclesare liposomes, biocompatible polymers, including natural polymers andsynthetic polymers; lipoproteins; polypeptides; polysaccharides;lipopolysaccharides; artificial viral envelopes; metal particles; andbacteria, viruses, such as baculovirus, adenovirus and retrovirus,bacteriophage, cosmid, plasmid, fungal vectors and other recombinationvehicles typically used in the art which have been described forexpression in a variety of eukaryotic and prokaryotic hosts. In someembodiments, the nucleic acid delivery vehicle is an expression vectorsuch as a plasmid. The vector may include any element to establish aconventional function of an expression vector, for example, a promoter,ribosome binding element, terminator, enhancer, selection marker, andorigin of replication. The promoter can be a constitutive, inducible orrepressible promoter. Exemplary promoters include, but are not limitedto, the cytomegalovirus (CMV) immediate early promoter, the RSV LTR, theMoMLV LTR, the phosphoglycerate kinase-1 (PGK) promoter, a simian virus40 (SV40) promoter and a CK6 promoter, a transthyretin promoter (TTR), aTK promoter, a tetracycline responsive promoter (TRE), an HBV promoter,an hAAT promoter, a LSP promoter, chimeric liver-specific promoters(LSPs), the E2F promoter, the telomerase (hTERT) promoter; thecytomegalovirus enhancer/chicken beta-actin/Rabbit β-globin promoter(CAG promoter; Niwa et al., Gene, 1991, 108(2):193-9) and the elongationfactor 1-alpha promoter (EF1-alpha) promoter (Kim et al., Gene, 1990,91(2):217-23 and Guo et al., Gene Ther., 1996, 3(9):802-10). A number ofexpression vectors capable of delivering nucleic acids to a cell (e.g.,bacterial cell, yeast cell, plant cell, or mammalian cell) are known inthe art and may be used herein for production of a fusion protein orfusion protein component in the cell. For example, E. coli can be usedto produce a fusion protein if transformed with a plasmid, such aspBR322 (Mandel et al., J. Mol. Biol., 1970, 53:154), engineered tocomprise a nucleic acid encoding the fusion protein. Expressed fusionproteins or fusion protein components can be harvested from the cellsand purified according to conventional techniques known in the art andas described herein.

Host Cells

Provided herein are host cells comprising a nucleic acid encoding afusion protein described herein. Nucleic acids encoding fusion proteinsor fusion protein components (e.g., an extracellular portion of a PDGFR,an extracellular portion of a VEGFR, and/or a multimerization domain)can be provided to a target cell by any means known in the art. In someembodiments, the nucleic acid encoding a protein of interest (e.g., afusion protein) is in a viral vector and the vector has been packaged,then the virions can be used to infect cells. In some embodiments, thenucleic acid encoding a protein of interest (e.g., a fusion protein) isin an expression vector such as a plasmid. Transfection ortransformation procedures as are appropriate for the particular cellscan be used for introducing a nucleic acid encoding a protein ofinterest (e.g., fusion protein) into a target cell. Formulationsutilizing polymers, liposomes, or nanospheres can be used for deliveryof nucleic acids encoding a protein of interest (e.g., a fusionprotein). Cells which can be transformed or transfected with recombinantconstructs according to the invention may be any which are convenient toone of skill in the art. Exemplary cell types which may be used includebacteria, yeast, fungi, insect, plant, and mammalian cells. Exemplarymammalian cells which may be used include, but are not limited to,fibroblasts, hepatocytes, endothelial cells, stem cells, hematopoieticcells, epithelial cells, myocytes, neuronal cells, and keratinocytes.Additional exemplary mammalian cell lines that can be used include, butare not limited to, COS cells, VERO cells, HeLa cells, Chinese hamsterovary (CHO) cells, 293 cells, NSO cells, SP20 cells, 3T3 fibroblastcells, W138 cells, BHK cells, HEPG2 cells, DUX cells and MDCK cells.These cells can be used to produce and harvest the protein of interest.In some embodiments, transformed or transfected cells can be provided toa cell or mammalian host. Suitable cells for delivery to a cell ormammalian host include any mammalian cell type from any organ, tumor, orcell line. For example, human, murine, goat, ovine, bovine, dog, cat,and porcine cells can be used.

The term “host cell” includes a cell which has been or can be arecipient for a vector(s) of this invention and the progeny thereof. Theprogeny may not necessarily be completely identical (in morphology or ingenomic of total DNA complement) to the original parent cell due tonatural, accidental, or deliberate mutation. Host cells are preferablyeukaryotic cells, preferably mammalian cells, most preferably humancells.

V. Methods of Producing Fusion Proteins and Fusion Protein Components

Provided herein are methods for producing fusion proteins or fusionprotein components (e.g., an extracellular portion of a PDGFR, anextracellular portion of a VEGFR, and/or a multimerization domain) ofthe invention as disclosed herein. In some aspects, a method is providedfor producing any fusion protein as disclosed herein comprisingculturing a host cell comprising a nucleic acid encoding any of thefusion proteins disclosed herein under a condition that produces thefusion protein, and recovering the fusion protein produced by the hostcell. In some embodiments, a nucleic acid encoding a fusion protein isselected from the group consisting of SEQ ID NOs:18-24.

(1) Culturing the Host Cells

Cells used to produce the fusion proteins or fusion protein components(e.g., an extracellular portion of a PDGFR, an extracellular portion ofa VEGFR, and/or a multimerization domain) of the invention are grown inmedia known in the art and suitable for culture of the selected hostcells. Examples of suitable media include Ham's FlO (Sigma), MinimalEssential Medium (MEM, Sigma), RPMI 1640 (Sigma), Dulbecco's ModifiedEagle's Medium (DMEM, Sigma), and Luria Broth (LB). In addition, any ofthe media described in Ham et al., Meth. Enz. 58:44 (1979), Barnes etal., Anal. Biochem. 102:255 (1980), U.S. Pat. Nos. 4,767,704; 4,657,866;4,927,762; 4,560,655; or 5,122,469; WIPO Publication Nos. WO 90/03430;WO 87/00195; or U.S. Pat. Re. 30,985 may be used as culture media forthe cells. A given medium is generally supplemented as necessary withhormones and/or other growth factors (such as insulin, transferrin, orepidermal growth factor), DHFR, salts (such as sodium chloride, calcium,magnesium, and phosphate), buffers (such as HEPES), nucleosides (such asadenosine and thymidine), antibiotics, trace elements, and glucose or anequivalent energy source. Any other necessary supplements may also beincluded at appropriate concentrations that would be known to thoseskilled in the art. The culture conditions, such as temperature, pH, andthe like, are those previously used with the cell selected forexpression, and will be apparent to one of skill in the art. For E. coligrowth, for example, the preferred temperature ranges from about 20° C.to about 39° C., more preferably from about 25° C. to about 37° C., evenmore preferably at about 30° C. The pH of the medium may be any pHranging from about 5 to about 9, depending mainly on the host organism.For E. coli, the pH is preferably from about 6.8 to about 7.4, and morepreferably about 7.0. If an inducible promoter is used in the expressionvector, protein expression is induced under conditions suitable for theactivation of the promoter. For example, if a PhoA promoter is used forcontrolling transcription, the transformed host cells may be cultured ina phosphate-limiting medium for induction. A variety of other inducersmay be used, according to the vector construct employed, as is known inthe art.

(2) Purification of Fusion Proteins or Fusion Protein Components

When using recombinant techniques, the fusion proteins or fusion proteincomponents (e.g., an extracellular portion of a PDGFR, an extracellularportion of a VEGFR, and/or a multimerization domain) described hereincan be produced intracellularly, in the periplasmic space, or secreteddirectly into the medium. If the polypeptides are producedintracellularly, as a first step, protein recovery typically involvesdisrupting the cell, generally by such means as osmotic shock,sonication or lysis. Once cells are disrupted, particulate debris fromeither host cells or lysed fragments is removed, for example, bycentrifugation or ultrafiltration. Where the polypeptides are secretedinto the medium, supernatants from such expression systems are generallyfirst filtered and concentrated using a commercially available proteinconcentration filter, for example, an Amicon or Millipore Pelliconultrafiltration unit. A protease inhibitor such as PMSF may be includedin any of the foregoing steps to inhibit proteolysis and antibiotics maybe included to prevent the growth of adventitious contaminants.

Compositions of fusion proteins or fusion protein components (e.g., anextracellular portion of a PDGFR, an extracellular portion of a VEGFR,and/or a multimerization domain) prepared from such cells can bepurified using, for example, hydroxylapatite chromatography, gelelectrophoresis, dialysis, and affinity chromatography. In someembodiments, protein A or protein G is used as an affinity ligand foruse in affinity chromatography. The suitability of protein A as anaffinity ligand depends on the species and isotype of any immunoglobulinFc region that is present in the fusion proteins (Lindmark et al., J.Immunol. Meth. 62:1-13 (1983). In some embodiments, protein A is used asan affinity ligand for isolating and purifying fusion proteins or fusionprotein components (e.g., an extracellular portion of a PDGFR, anextracellular portion of a VEGFR, and/or a multimerization domain) asdescribed herein. In some embodiments, protein G is used as an affinityligand for isolating and purifying fusion proteins or fusion proteincomponents (e.g., an extracellular portion of a PDGFR, an extracellularportion of a VEGFR, and/or a multimerization domain) as describedherein. The matrix to which the affinity ligand is attached is mostoften agarose, but other matrices are available. Mechanically stablematrices such as controlled pore glass or poly(styrene-divinyl)benzeneallow for faster flow rates and shorter processing times than can beachieved with agarose. Other techniques for protein purification, suchas fractionation on an ion-exchange column, ethanol precipitation,Reverse Phase HPLC, chromatography on silica, heparin, SEPHAROSE™, oranion or cation exchange resins (such as a polyaspartic acid column), aswell as chromatofocusing, SDS-PAGE, and ammonium sulfate precipitationare also available depending on the fusion proteins or fusion proteincomponents (e.g., an extracellular portion of a PDGFR, an extracellularportion of a VEGFR, and/or a multimerization domain) to be recovered. Insome embodiments, the recovered fusion protein is substantially pure. Ina further embodiment, the recovered fusion protein is at least any of90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% pure. Following anypreliminary purification step or steps, the mixture comprising thefusion proteins or fusion protein components (e.g., an extracellularportion of a PDGFR, an extracellular portion of a VEGFR, and/or amultimerization domain) of interest and contaminants may be subjected tolow pH hydrophobic interaction chromatography using an elution buffer ata pH between about 2.5-4.5, preferably performed at low saltconcentrations (e.g., from about 0-0.25 M salt).

In general, various methodologies for preparing fusion proteins orfusion protein components (e.g., an extracellular portion of a PDGFR, anextracellular portion of a VEGFR, and/or a multimerization domain) foruse in research, testing, and clinical applications are well-establishedin the art, consistent with the above-described methodologies and/or asdeemed appropriate by one skilled in the art for a particular fusionproteins or fusion protein components of interest.

(3) Biological Activities of Fusion Proteins or Fusion ProteinComponents

Proteins may be purified and identified using commonly known methodssuch as fractionation on immunoaffinity or ion-exchange columns; ethanolprecipitation; reverse phase HPLC; chromatography on silica or on acation exchange resin such as DEAE; chromatofocusing; SDS-PAGE; ammoniumsulfate precipitation; gel filtration using, for example, Sephadex G-75;hydrophobic affinity resins, ligand affinity using a suitable bindingpartner immobilized on a matrix, centrifugation, ELISA, BIACore, Westernblot assay, amino acid and nucleic acid sequencing, and biologicalactivity.

The fusion proteins or fusion protein components disclosed herein may becharacterized or assessed for biological activities including, but notlimited to, affinity to a target binding partner (e.g., a PDGF and/orVEGF family protein), competitive binding (e.g., blocking of targetbinding partner to PDGFR or VEGFR), inhibitory activity (e.g.,inhibition of PDGF or VEGF pathway activation), inhibition of cellproliferation, inhibition of tumor growth, and inhibition ofangiogenesis (e.g., choroidal neovascularization). In some embodiments,the fusion proteins or fusion protein components disclosed herein can beassessed for biological activity in vivo or in vitro. In any of theassays described herein, the assay is performed at a temperature of 4°C., 20-28° C. (e.g., 25° C.), or 37° C.

The fusion proteins or fusion protein components disclosed herein can beassessed for affinity to a binding partner such as a PDGF family protein(e.g., PDGF-A, PDGF-B, PDGF-C, or PDGF-D), a dimer of a PDGF familyprotein (e.g., PDGF-AA, PDGF-AB, PDGF-BB, PDGF-CC, or PDGF-DD) or a VEGFfamily protein (e.g., VEGF-A VEGF-B, VEGF-C, VEGF-D or PlGF). Manymethods for assessing binding affinity are known in the art and can beused to identify the binding affinities of fusion proteins or fusionprotein components to a binding partner. Binding affinities can beexpressed as dissociation constant (Kd) values or half maximal effectiveconcentration (EC50) values. Techniques for determining bindingaffinities (e.g., Kd values) are well known in the art such asEnzyme-Linked Immunosorbent Assay (ELISA) and BIAcore. See Harlow andLane, Antibodies: A Laboratory Manual, CSH Publications, NY (1988);Ausubel et al., Current Protocols in Molecular Biology, John Wiley &Sons, New York, (2009); Altschuh et al., Biochem., 31:6298 (1992); andthe BIAcore method disclosed by Pharmacia Biosensor, all of which areincorporated herein by reference. For example, binding affinities of thefusion proteins to a binding partner can be determined using ELISA. Insome embodiments, binding of fusion proteins to PDGF-BB is assayed usingELISA. In this exemplary assay, secreted fusion proteins were seriallydiluted, mixed with human PDGF BB ligand at a 20 pM final concentrationand incubated overnight at room temperature on an orbital shakerplatform. After incubation, the amount of unbound PDGF-BB is measured bya human PDGF-specific ELISA (Human PDGF-BB DuoSet Product #DY220, R&DSystems). Statistical significance in binding affinities is analyzedusing Prism 5.0d (GraphPad Software, Inc) and was calculated using the2-way ANOVA test followed by Bonferroni correction. In a furtherexample, binding of a fusion protein to a VEGF family protein is assayedusing ELISA. In an exemplary assay, secreted fusion proteins areserially diluted, mixed with human VEGF at a 20 pM final concentrationand incubated overnight at room temperature on an orbital shakerplatform. The amount of unbound VEGF is then measured by a humanVEGF-specific ELISA (Human VEGF Quantikine ELISA kit Cat# DVE00, R&DSystems).

In any of the embodiments herein, a fusion protein has an EC50 of ≦1 μM,≦100 nM, ≦10 nM, ≦1 nM, ≦0.1 nM, ≦0.01 nM, or ≦0.001 nM (e.g., 10⁻⁸M orless, e.g., from 10⁻⁸M to 10⁻¹³M, e.g., from 10⁻⁹M to 10⁻¹³ M) forinhibition of an activity (e.g., inhibition of PDGF activity and/or VEGFactivity). In any of the embodiments herein, a fusion protein has a Kdfor a binding partner (e.g., PDGF and/or VEGF) of less than about any ofabout 1.0 mM, 500 μM, 100 μM, 50 μM, 25 μM, 10 μM, 5 μM, 1 μM, 900 nM,800 nM, 700 nM, 600 nM, 500 nM, 400 nM, 350 nM, 300 nM, 250 nM, 200 nM,150 nM, 100 nM, 95 nM, 90 nM, 85 nM, 80 nM, 75 nM, 70 nM, 65 nM, 60 nM,55 nM, 50 nM, 45 nM, 40 nM, 35 nM, 30 nM, 25 nM, 20 nM, 15 nM, 10 nM, 5nM, 1 nM, 900 pM, 800 pM, 700 pM, 600 pM, 500 pM, 400 pM, 300 pM, 200pM, 100 pM, 50 pM, 25 pM, 12.5 pM, 6.25 pM, 5 pM, 4 pM, or 3 pM,inclusive, including any values in between these numbers. In someembodiments, the fusion protein variants described herein bind to abinding partner with a higher affinity compared to the binding of awild-type fusion protein described herein. In some aspects, the fusionprotein variant binds to a binding partner with at least any of 10, 20,30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900,1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 6000, 7000, 8000,9000, or 10,000, inclusive, including any value in between thesenumbers, higher fold affinity compared to the binding of the bindingpartner by a fusion protein comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NOs:9-15.

In some embodiments, the fusion proteins disclosed herein can beassessed for anti-proliferative activities such as reduction of cellproliferation. Many methods for assessing anti-proliferative propertiesfor a fusion protein are known in the art. In one exemplary assay, humanumbilical vein endothelial cells (HUVECs) can be used to demonstrateinhibition of VEGF-dependent and/or PDGF-dependent cell proliferation bya fusion protein described herein. In this assay, the fusion protein isapplied to HUVECs in the presence of VEGF and/or PDGF and cellproliferation is measure. For example, HUVECs (HUVEC-Cambrex Bio ScienceWalkersville, Inc) are seeded in a 96 well plate at a density of 2,000cells/well in Media 199 (Invitrogen) supplemented with 5% Fetal BovineSerum (Invitrogen) and settled overnight. After incubation, the media isreplaced with Media 199 (Invitrogen) supplemented with 5% Fetal BovineSerum (Invitrogen) containing an equal volume (5 μl) of harvested cellculture and recombinant hVEGF-165 ligand alone at a final concentrationof 10 ng/ml (R&D Systems Cat#293-VE), or in combination with PDGF-BBligand at a final concentration of 20 ng/ml (R&D Systems Cat#220-BB) ina final volume of 100 μl per well. Cells are incubated at 37° C. in 5%CO₂ for three to four days. Cell Titer 96 AQ_(ueous) One SolutionReagent (Promega Cat# G3580) is added at 20 μl/well and absorbance at490 nm is taken four hours later to determine inhibition of cellproliferation by the fusion protein. In some embodiments,anti-angiogenic properties for a fusion protein are measured usingtechniques well known in the art. In an exemplary assay, an animal modelof wet age-related macular degeneration is used to assay inhibition ofneovascularization in the eye by the fusion protein. In this assay, theeyes of normal adult mouse is treated with a single intravitrealinjection of a fusion protein or an rAAV particle comprising a nucleicacid encoding a fusion protein into the left eye (OS) on study day 0while the right eye (OD) is left naïve to treatment. CNV is induced inboth eyes using a laser (e.g., 3 burns placed per eye. 200 mW power, 50μm spot, 100 ms) on study day 28. Mice are perfused with FITC-Dextranand euthanized on study day 42. The eyes are collected, fixed in 10%neutral buffered formalin and choroidal flatmounts are subsequentlyprepared in order to examine the extent of neovascularization. Thenumber of burns without CNV in the treated (OS) eye is compared to thecontralateral (OD) eye to determine the efficacy of the fusion protein.See, e.g., Example 5.

VI. Viral Particles and Methods of Producing Viral Particles

Also provided herein are viral particles comprising a nucleic acidencoding a fusion protein described herein. Viral vectors can be usedfor delivery of a nucleic acid encoding a fusion protein or fusionprotein component for expression of the protein in a target cell withina particular target tissue (e.g., a diseased tissue). Many species ofvirus are known, and many have been studied for purposes of deliveringnucleic acids to target cells. The exogenous nucleic acid can beinserted into a vector such as adenovirus, partially-deleted adenovirus,fully-deleted adenovirus, adeno-associated virus (AAV), retrovirus,lentivirus, and so forth for delivery to a cell. In some embodiments,the cell is in an individual and the virus is delivered via anintravenous, intramuscular, intraportal or other route ofadministration. The most commonly used viral vectors include thosederived from adenoviruses, adeno-associated viruses (AAV) andretroviruses, including lentiviruses, such as human immunodeficiencyvirus (HIV). For exemplary viral vectors see U.S. Pat. No. 7,928,072 andWO2006/113277, both of which are incorporated herein by reference intheir entirety.

In some embodiments, the viral particle is a recombinant AAV particlecomprising a nucleic acid comprising one or two AAV ITRs and a sequenceencoding a fusion protein described herein flanked by one or two ITRs.The nucleic acid is encapsidated in the AAV particle. The AAV particlealso comprises capsid proteins. In some embodiments, the nucleic acidcomprises operatively linked components in the direction oftranscription, control sequences including transcription initiation andtermination sequences, and the protein coding sequence(s) of interest(e.g., nucleic acid encoding a fusion protein). These components areflanked on the 5′ and 3′ end by functional AAV ITR sequences. By“functional AAV ITR sequences” it is meant that the ITR sequencesfunction as intended for the rescue, replication and packaging of theAAV virion. See Davidson et al., PNAS, 2000, 97(7)3428-32; Passini etal., J. Virol., 2003, 77(12):7034-40; and Pechan et al., Gene Ther.,2009, 16:10-16, all of which are incorporated herein in their entiretyby reference. For practicing some aspects of the invention, therecombinant vectors comprise at least all of the sequences of AAVessential for encapsidation and the physical structures for infection bythe rAAV. AAV ITRs for use in the vectors of the invention need not havea wild-type nucleotide sequence (e.g., as described in Kotin, Hum. GeneTher., 1994, 5:793-801), and may be altered by the insertion, deletionor substitution of nucleotides or the AAV ITRs may be derived from anyof several AAV serotypes. More than 40 serotypes of AAV are currentlyknown, and new serotypes and variants of existing serotypes continue tobe identified. See Gao et al., PNAS, 2002, 99(18): 11854-6; Gao et al.,PNAS, 2003, 100(10):6081-6; and Bossis et al., J. Virol., 2003,77(12):6799-810. Use of any AAV serotype is considered within the scopeof the present invention. In some embodiments, a rAAV vector is a vectorderived from an AAV serotype, including without limitation, AAV 1, AAV2,AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10. In someembodiments, the nucleic acid in the AAV comprises an ITR of AAV1, AAV2,AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10. In someembodiments, a nucleic acid encoding a fusion protein selected from thegroup consisting of SEQ ID NOs:12-15 is flanked by at least one AAV ITR.In some embodiments, the nucleic acid is selected from the groupconsisting of SEQ ID Nos:21-24. In further embodiments, the rAAVparticle comprises capsid proteins of AAV1, AAV2, AAV3, AAV4, AAV5, AA6,AAV7, AAV8, AAV9, AAVrh.8, or AAVrh.10.

Different AAV serotypes are used to optimize transduction of particulartarget cells or to target specific cell types within a particular targettissue (e.g., a diseased tissue). A rAAV particle can comprise viralproteins and viral nucleic acids of the same serotype or a mixedserotype. For example, a rAAV particle can comprise AAV2 capsid proteinsand at least one AAV2 ITR or it can comprise AAV2 capsid proteins and atleast one AAV1 ITR. In another example, a rAAV particle can compriseAAV1 capsid proteins and at least one AAV2 ITR. In yet another example,a rAAV particle can comprise capsid proteins from both AAV1 and AAV2,and further comprise at least one AAV2 ITR. Any combination of AAVserotypes for production of a rAAV particle is provided herein as ifeach combination had been expressly stated herein.

The rAAV particles can be produced using methods know in the art. See,e.g., U.S. Pat. Nos. 6,566,118, 6,989,264, 6,995,006. In practicing theinvention, host cells for producing rAAV particles include mammaliancells, insect cells, plant cells, microorganisms and yeast. Host cellscan also be packaging cells in which the AAV rep and cap genes arestably maintained in the host cell or producer cells in which the AAVvector genome is stably maintained. Exemplary packaging and producercells are derived from 293, A549 or HeLa cells. AAV vectors are purifiedand formulated using standard techniques known in the art.

In some aspects, a method is provided for producing any rAAV particle asdisclosed herein comprising (a) culturing a host cell under a conditionthat rAAV particles are produced, wherein the host cell comprises (i)one or more AAV package genes, wherein each said AAV packaging geneencodes an AAV replication or encapsidation protein; (ii) an rAAVpro-vector comprising a nucleic acid encoding any fusion proteindisclosed herein flanked by at least one AAV ITR, and (iii) an AAVhelper function; and (b) recovering the rAAV particles produced by thehost cell. In some embodiments, a nucleic acid encodes a fusion proteinselected from the group consisting of SEQ ID NOs:12-15. In someembodiments, said at least one AAV ITR is selected from the groupconsisting of AAV1, AAV2, AAV3, AAV4, AAV5, AA6, AAV7, AAV8, AAV9,AAVrh.8, and AAVrh.10 ITR. In some embodiments, said encapsidationprotein is selected from the group consisting of AAV1, AAV2, AAV3, AAV4,AAV5, AA6, AAV7, AAV8, AAV9, AAVrh.8, and AAVrh.10 capsid protein. In afurther embodiment, the rAAV particles are purified. The term “purified”as used herein includes a preparation of rAAV particles devoid of atleast some of the other components that may also be present where therAAV particles naturally occur or are initially prepared from. Thus, forexample, isolated rAAV particles may be prepared using a purificationtechnique to enrich it from a source mixture, such as a culture lysateor production culture supernatant. Enrichment can be measured in avariety of ways, such as, for example, by the proportion ofDNase-resistant particles (DRPs) present in a solution, or byinfectivity, or it can be measured in relation to a second, potentiallyinterfering substance present in the source mixture, such ascontaminants, including production culture contaminants or in-processcontaminants, including helper virus, media components, and the like.

Also provided herein are pharmaceutical compositions comprising a rAAVparticle comprising a nucleic acid encoding a fusion protein of theinvention and a pharmaceutically acceptable carrier. The pharmaceuticalcompositions may be suitable for a variety of modes of administrationdescribed herein, including for example systemic or localizedadministration. A pharmaceutical composition of a rAAV comprising anucleic acid encoding a fusion protein described herein can beintroduced systemically, e.g., by intravenous injection, by catheter,see U.S. Pat. No. 5,328,470, or by stereotactic injection, Chen et al.,1994, PNAS, 91: 3054-3057. The pharmaceutical compositions can be in theform of eye drops, injectable solutions, or in a form suitable forinhalation or oral administration. In some embodiments, thepharmaceutical compositions comprising a fusion protein described hereinand a pharmaceutically acceptable carrier is suitable for administrationto human. In some embodiments, the pharmaceutical compositionscomprising a fusion protein described herein and a pharmaceuticallyacceptable carrier is suitable for intravitreal injection or topicalapplication to the eye. Such pharmaceutically acceptable carriers can besterile liquids, such as water and oil, including those of petroleum,animal, vegetable or synthetic origin, such as peanut oil, soybean oil,mineral oil, and the like. Saline solutions and aqueous dextrose,polyethylene glycol (PEG) and glycerol solutions can also be employed asliquid carriers, particularly for injectable solutions. Thepharmaceutical composition may further comprise additional ingredients,for example preservatives, buffers, tonicity agents, antioxidants andstabilizers, nonionic wetting or clarifying agents, viscosity-increasingagents, and the like. The pharmaceutical compositions described hereincan be packaged in single unit dosages or in multidosage forms. Thecompositions are generally formulated as sterile and substantiallyisotonic solution. Compositions can also be formulated to have osmoticvalues that are compatible with the aqueous humor of the eye andophthalmic tissues. Such osmotic values will generally be in the rangeof from about 200 to about 400 mOsm/kg, but will preferably be about 300mOsm/kg. Ophthalmic solutions useful for storing and/or deliveringexpression vectors or viral vectors have been disclosed, for example, inWO03077796A2.

VII. Methods of Treatment Using Fusion Proteins and Viral Particles

The methods of the present invention use any fusion protein disclosedherein. In some embodiments, the fusion protein binds a PDGF protein ora VEGF protein. In some embodiments, the fusion protein binds a PDGFRprotein and a VEGFR protein. The fusion proteins described herein mayhave one or more of the following characteristics: (a) bind one or moreproteins of the PDGF family such as PDGF-A, PDGF-B, PDGF-C, or PDGF-D;(b) bind one or more proteins of the VEGF family such as VEGF-A, VEGF-B,VEGF-C, VEGF-D, or PlGF; (c) block binding of a PDGF family protein to aPDGF receptor; (d) block binding of a VEGF family protein to a VEGFreceptor; (e) inhibit activation of the PDGF signaling pathway and/orVEGF signaling pathway; (f) treat and/or prevent a disease such as anocular disease, autoimmune disease, inflammatory disease, or cancer. Theactivities of fusion proteins may be measured in vitro and/or in vivo.

The present invention provides methods of treating a disease (such as anocular disease, an inflammatory disease, an autoimmune disease, orcancer) by administering an effective amount of any fusion proteindescribed herein to an individual. In some embodiments, a method oftreating a disease comprises administering an effective amount of acomposition comprising the fusion protein to an individual. In someembodiments, a method of treating a disease comprises administering aneffective amount of a rAAV comprising a nucleic acid encoding the fusionprotein to an individual. Methods of treating or preventing one or moreaspects or symptoms of a disease (such as an ocular disease, aninflammatory disease, an autoimmune disease, or cancer) by administeringan effective amount of any fusion protein described herein to anindividual are also provided. In some embodiments, a method of treatingor preventing one or more aspects or symptoms of a disease comprisesadministering an effective amount of a composition comprising the fusionprotein to an individual. In some embodiments, a method of treating orpreventing one or more aspects or symptoms of a disease comprisesadministering an effective amount of a rAAV comprising a nucleic acidencoding the fusion protein to an individual.

The methods described herein can be used for the treatment of a varietyof diseases, including, but not limited to, inflammatory disease, oculardisease, autoimmune disease, or cancer. In some embodiments, the diseaseto be treated includes, but is not limited to, rheumatoid arthritis,inflammatory arthritis, osteoarthritis, cancer, age-related maculardegeneration (AMD) (such as wet AMD or dry AMD), ocular diseasecharacterized by neovascularization (such as choroidalneovascularization), uveitis (such as anterior uveitis or posterioruveitis), retinitis pigmentosa, and diabetic retinopathy.

In certain embodiments, the methods and compositions of the inventioncan be used to treat an autoimmune disease. In some embodiments, theautoimmune disease is rheumatoid arthritis, multiple sclerosis, orsystemic lupus erythematosus. Rheumatoid arthritis (RA) is a chronicautoimmune disease that leads to inflammation of the joints. While RAprincipally affects synovial joints, it can affect surrounding tissuesand organs. The pathology of RA involves an inflammatory process thatcan lead to the destruction of cartilage and the ankylosis (fusion) ofjoints. Other pathological manifestations of RA include vasculitis(inflammation of the blood vessels), which can affect nearly any organsystem and can cause additional complications, including polyneuropathy,cutaneous ulceration, and visceral infarction. Pleuropulmonarymanifestations include pleuritis, interstitial fibrosis, Caplan'ssyndrome, pleuropulmonary nodules, pneumonitis, rheumatoid lung diseaseand arteritis. Other manifestations include the development ofinflammatory rheumatoid nodules on a variety of periarticular structuressuch as extensor surfaces, as well as on pleura and meninges. Weaknessand atrophy of skeletal muscle are common.

In certain embodiments, the methods and compositions of the inventioncan be used to treat an inflammatory disease. In some embodiments, theinflammatory disease is inflammatory arthritis, osteoarthritis,psoriasis, chronic inflammation, irritable bowel disease, lunginflammation or asthma. Inflammatory arthritis refers to inflammation ofthe joints that can result from an autoimmune disease such as, e.g.,ankylosing spondylitis, juvenile idiopathic arthritis, mixed connectivetissue disease, psoriatic arthritis, reactive arthritis, scleroderma,Sjogren's Syndrome, Still's Disease, and systemic lupus erythematosus.Inflammatory arthritis can also be caused by certain types of bacteria(such as with reactive arthritis) or by deposits of crystallinestructures in the joints (such as with gout and pseudogout). Thecharacteristic symptoms of inflammatory arthritis are pain and swellingof one or more joints, which may be warmer than the other joints.Stiffness of the joints following prolonged inactivity (such as in themorning or after sitting for a length of time) is a very common symptom.Patients with inflammatory arthritis usually have multiple jointcomplaints. Osteoarthritis, also known as degenerative arthritis ordegenerative joint disease, is a group of mechanical abnormalitiesinvolving degradation of joints, including articular cartilage andsubchondral bone. Symptoms may include joint pain, tenderness,stiffness, locking, and sometimes an effusion (i.e., the presence ofincreased intra-articular fluid). A variety of causes, e.g., hereditary,developmental, metabolic, obesity-related, and mechanical, may initiateprocesses leading to loss of cartilage. As breakdown products from thecartilage are released into the synovial space, the cells lining thejoint attempt to remove them. New bone outgrowths, or “spurs” can form.Often, when bone becomes less well protected by cartilage, bone may beexposed and damaged. These bone changes, in combination withinflammation of the joint, cause pain. As a result of decreased movementsecondary to pain, regional muscles may atrophy, and ligaments maybecome more lax.

Persistent and unregulated angiogenesis occurs in a multiplicity ofdisease states such as cancer. In cancer, cells divide and growuncontrollably, forming malignant tumors, which vascularize and invadenearby parts of the body. The cancer may also spread (metastasize) tomore distant parts of the body through the lymphatic system orbloodstream. The causes of cancer can be environmental (due to exposureto chemicals, radiation or due to lifestyle), hereditary, or infectious.In some embodiments, the methods and compositions of the invention canbe used to treat cancer. In some embodiments, the cancer is prostatecancer, breast cancer, lung cancer, esophageal cancer, colon cancer,rectal cancer, liver cancer, urinary tract cancer (e.g., bladdercancer), kidney cancer, lung cancer (e.g., non-small cell lung cancer),ovarian cancer, cervical cancer, endometrial cancer, pancreatic cancer,stomach cancer, thyroid cancer, skin cancer (e.g., melanoma),hematopoietic cancers of lymphoid or myeloid lineage, head and neckcancer, nasopharyngeal carcinoma (NPC), glioblastoma, teratocarcinoma,neuroblastoma, adenocarcinoma, cancers of mesenchymal origin such as afibrosarcoma or rhabdomyosarcoma, soft tissue sarcoma and carcinoma,choriocarcinioma, hepatoblastoma, Karposi's sarcoma or Wilm's tumor.

Other diseases that are associated with angiogenesis can be treated withthe methods and compositions disclosed herein. These diseases includeatherosclerosis, retrolentral fibroplasia, thyroid hyperplasias(including grave's disease), nephrotic syndrome, preclampasia, ascites,pericardial effusion (such as associated with pericarditis) and pleuraleffusion.

In some embodiments, the methods and compositions of the invention canbe used to treat an ocular disease. In some embodiments, the oculardisease is AMD such as wet AMD or dry AMD, uveitis, retinitispigmentosa, neovascular glaucoma, diabetic retinopathy, and other eyediseases that involve a local inflammatory process. In some embodiments,the ocular disease is characterized by neovascularization, such aschoroidal neovascularization. In some embodiments, the ocular disease isa result of corneal transplantation. In some embodiments, the inventionprovides methods of treating or preventing one or more aspects orsymptoms of an ocular disease including, but not limited to, formationof ocular drusen, inflammation in the eye or eye tissue and loss ofvision. In certain embodiments, the compositions and methods describedherein can be used to detect and/or treat uveitis, i.e., inflammation ofthe uvea, the middle layer of the eye beneath the sclera. Uveitis isestimated to be responsible for approximately 10%-20% of the blindnessin the United States. The uvea is traditionally divided into 3 areas,from front to back, the iris, ciliary body, and choroid. The primefunctions of the uvea are nutrition and gas exchange, light absorption,and secretion of the aqueous humour by the cilliary processes. Uveitisis typically associated with exposure to toxins, infection, and/orautoimmune disorders. However, in many cases, the cause is unknown.Uveitis can affect one or both eyes. Symptoms may develop rapidly andcan include blurred vision, floating dark spots in the field of vision,eye pain, eye redness, and sensitivity to light. The most common form ofuveitis is anterior uveitis, or iritis, which involves inflammation ofthe iris. Pars plantis refers to inflammation of the uvea in the middleof the eye, i.e., between the iris and the choroid. Posterior uveitisaffects the back of the eye, i.e., the choroid. Inflammation associatedwith posterior uveitis can also affect the retina (retinitis) or theblood vessels at the back of the eye (vasculitis).

In certain embodiments, the methods and compositions of the inventioncan be used to treat retinitis pigmentosa (RP). RP is a heritable eyedisease that is caused by abnormalities of the photoreceptors (rods andcones) or the retinal pigment epithelium of the retina. The disease canlead to progressive sight loss and often blindness. The symptoms of RPinclude decreased vision at night or in low light, loss of side(peripheral) vision, and, in advanced cases, loss of central vision. Thediagnosis of RP relies upon the documentation of progressive loss inphotoreceptor cell function via visual field testing andelectroretinography. At least 35 genetic loci are known to cause“non-syndromic retinitis pigmentosa” (i.e., RP that is not the result ofanother disease or part of a wider syndrome).

In certain embodiments, the methods and compositions of the inventioncan be used to treat diabetic retinopathy. Diabetic retinopathy refersto damage to the retina caused by the complications of diabetes.Specifically, vascular walls are compromised by hyperglycemia, changingthe formation of the blood-retinal barrier and making the retinal bloodvessels more permeable. The damaged blood vessels least fluid and lipidsinto the macula, causing the macular to swell (i.e., macular edema),which blurs vision. As the disease progresses, it enters a proliferativestage, in which blood vessels grow along the retina and in the vitreoushumour that fills the eye. These blood vessels can bleed, cloud vision,and e.g., destroy the retina, cause retinal detachment, or causeneovascular glaucoma.

In certain embodiments, the methods and compositions of the inventioncan be used to treat age-related macular degeneration (AMD). AMD ischaracterized by progressive loss of central vision which occurs as aresult of damage to the photoreceptor cells in an area of the retinacalled the macula. AMD has been broadly classified into two clinicalstates: a wet form and a dry form, with the dry form making up to 80-90%of total cases. Dry AMD is characterized by the formation of maculardrusen, tiny yellow or white accumulations of extracellular materialthat builds up between Bruch's membrane and the retinal pigmentepithelium of the eye. Wet AMD, which accounts for approximately 90% ofserious vision loss, is associated with neovascularization, whereinblood vessels grow up from the choroid beneath the retina, and with theleakage of these new vessels. The accumulation of blood and fluid cancause retinal detachment followed by rapid photoreceptor degenerationand loss of vision in either form of AMD. It is generally accepted thatthe wet form of AMD is preceded by and arises from the dry form.

Methods of delivering an effective amount of a fusion protein to asubject are provided herein. The fusion protein can be delivered to asubject in a composition. The fusion protein can also be delivered to asubject by a rAAV comprising a nucleic acid encoding the fusion protein.Compositions comprising the fusion protein or the rAAV comprising anucleic acid encoding the fusion protein are contemplated herein.

The compositions described herein can be administered to an individualvia any route, including, but not limited to, intravenous (e.g., byinfusion pumps), intraperitoneal, intraocular, intra-arterial,intrapulmonary, oral, inhalation, intravesicular, intramuscular,intra-tracheal, subcutaneous, intraocular, intrathecal, transdermal,transpleural, intraarterial, topical, inhalational (e.g., as mists ofsprays), mucosal (such as via nasal mucosa), subcutaneous, transdermal,gastrointestinal, intraarticular, intracisternal, intraventricular,intracranial, intraurethral, intrahepatic, and intratumoral. In someembodiments, the compositions are administered intravascularly, such asintravenously (IV) or intraarterially. In some embodiments, thecompositions are administered directly into arteries. In someembodiments, the compositions are administered systemically (for exampleby intravenous injection). In some embodiments, the compositions areadministered locally (for example by intraarterial or intraocularinjection).

In some embodiments, the compositions are administered directly to theeye or the eye tissue. In some embodiments, the compositions areadministered topically to the eye, for example, in eye drops. In someembodiments, the compositions are administered by injection to the eye(intraocular injection) or to the tissues associated with the eye. Thecompositions can be administered, for example, by intraocular injection,periocular injection, subretinal injection, intravitreal injection,trans-septal injection, subscleral injection, intrachoroidal injection,intracameral injection, subconjunctival injection, sub-Tenon'sinjection, retrobulbar injection, peribulbar injection, or posteriorjuxtascleral delivery. These methods are known in the art. For example,for a description of exemplary periocular routes for retinal drugdelivery, see Raghava et al., Expert Opin. Drug Deliv., 2004,1(1):99-114. The compositions may be administered, for example, to thevitreous, aqueous humor, sclera, conjunctiva, the area between thesclera and conjunctiva, the retina choroids tissues, macula, or otherarea in or proximate to the eye of an individual. The compositions canalso be administered to the individual as an implant. Preferred implantsare biocompatible and/or biodegradable sustained release formulationswhich gradually release the compounds over a period of time. Ocularimplants for drug delivery are well-known in the art. See, e.g., U.S.Pat. Nos. 5,501,856, 5,476,511, and 6,331,313. The compositions can alsobe administered to the individual using iontophoresis, including, butare not limited to, the ionophoretic methods described in U.S. Pat. No.4,454,151 and U.S. Pat. App. Pub. No. 2003/0181531 and 2004/0058313.

The optimal effective amount of the compositions can be determinedempirically and will depend on the type and severity of the disease,route of administration, disease progression and health, mass and bodyarea of the individual. Such determinations are within the skill of onein the art. For example, when administered intraocularly, the amount ofa rAAV comprising a nucleic acid encoding a fusion protein describedherein can be administered to an individual as a DNAse particleresistant (drps) titer of about 10⁴ to about 10¹⁴ drps per dose. In someembodiments, the amount of a rAAV comprising a nucleic acid encodingfusion protein can be administered to an individual at about 10⁵ toabout 10¹³, about 10⁶ to about 10¹², about 10⁷ to about 10¹¹, about 10⁸to about 10¹⁰, about 10⁹ to about 10¹⁰, about 10¹⁰ to about 10¹¹, orabout 10¹¹ to about 10¹² drps per dose.

Compositions comprising a fusion protein may be administered in a singledaily dose, or the total daily dose may be administered in divideddosages of two, three, or four times daily. Compositions comprising afusion protein can also be administered six times a week, five times aweek, four times a week, three times a week, twice a week, once a week,once every two weeks, once every three weeks, once a month, once everytwo months, once every three months, once every six months, once everynine months, or once every year. Compositions comprising a rAAVcomprising a nucleic acid encoding a fusion protein can be administeredless frequently, for example, once every three months, every fourmonths, once every five months, once every six months, once every sevenmonths, once every eight months, once every nine months, once every tenmonths, once every eleven months, or once every year. In someembodiments, a single dose of a composition comprising a rAAV comprisinga nucleic acid encoding a fusion protein described herein isadministered once a year. The compositions may also be administered in asustained release formulation, such as in an implant which graduallyreleases the composition for use over a period of time, and which allowsfor the composition to be administered less frequently, such as once amonth, once every 2-6 months, once every year, or even a singleadministration. The sustained release devices (such as pellets,nanoparticles, microparticles, nanospheres, microspheres, and the like)may be administered by injection or surgical implanted in variouslocations in the eye or tissue associated with the eye, such asintraocular, intravitreal, subretinal, periocular, subconjunctival, orsub-Tenons.

Compositions of the invention (e.g., a fusion protein or a rAAVcomprising a nucleic acid encoding a fusion protein) can be used eitheralone or in combination with one or more additional therapeutic agents.For example, the compositions of the invention can be administered aloneor in combination with other therapeutic agents known to have abeneficial effect on age-related macular degeneration (AMD), retinalattachment or damaged retinal tissue. Exemplary therapeutic agentsinclude complement inhibitors, anti-angiogenics, anti-VEGF agents(including, but not limited to Macugen (pegaptanib sodium), Eylea (VEGFTrap-Eye), and anti-VEGF antibody, such as Lucentis® or Avastin®), andanti-PDGF agents (such as Fostiva™). The compositions of the inventioncan be administered in combination with nutritional supplements shown tobe beneficial in lowering the risk of macular degeneration progressingto advanced stages, e.g., vitamin C, vitamin E, beta carotene, zincoxide, and copper. Other useful cofactors include symptom-alleviatingcofactors, including antiseptics, antibiotics, antiviral and antifungalagents, and analgesics and anesthetics. In some embodiments, acombination is provided as a simultaneous administration, wherein afusion protein or a rAAV comprising a nucleic acid encoding a fusionprotein and at least one therapeutic agent is administered together inthe same composition or administered simultaneously in differentcompositions. In some embodiments, a combination is provided as aseparate administration, wherein the administration of a fusion proteinor a rAAV comprising a nucleic acid encoding a fusion protein can occurprior to, simultaneously, and/or following administration of at leastone therapeutic agent. The interval between sequential administrationcan be in terms of at least (or, alternatively, less than) minutes,hours, or days.

The compositions described herein can also be used in conjunction withother AMD therapies, such as photodynamic therapy. Photodynamic therapyentails the intravenous administration of Visudyne (verteporfin), afterwhich light of a specific wavelength is applied to the abnormal bloodvessels. The light activates the Visudyne and obliterates the vessels.Alternatively, the compositions described herein can be used inconjunction with laser therapy, which entails using a high-energy laserbeam to destroy abnormal blood vessels under the macula.

VIII. Articles of Manufacture and Kits

Also provided are kits or articles of manufacture comprising thecompositions described herein (e.g., fusion proteins or rAAV particles)in suitable packaging. Suitable packaging for compositions (such asophthalmic compositions) described herein are known in the art, andinclude, for example, vials (such as sealed vials), vessels, ampules,bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags),and the like. These articles of manufacture may further be sterilizedand/or sealed.

The present invention also provides kits comprising compositionsdescribed herein and may further comprise instruction(s) on methods ofusing the composition, such as uses described herein. The kits describedherein may further include other materials desirable from a commercialand user standpoint, including other buffers, diluents, filters,needles, syringes, and package inserts with instructions for performingany methods described herein. For example, in some embodiments, the kitcomprises a fusion protein described herein and/or a rAAV encoding afusion protein described herein, a pharmaceutically acceptable carriersuitable for intraocular injection, and one or more of: a buffer, adiluent, a filter, a needle, a syringe, and a package insert withinstructions for performing intraocular injection.

EXAMPLES Example 1 Production of sPDGFR-β/Fc Fusion Proteins

The PDGF-beta receptor (PDGFR-β) ectodomain contains 5 extracellulardomains (ECD) numbered 1 to 5 from N-terminus to C-terminus of theprotein. The full length PDGFR-β ectodomain was used to generate severaltruncated soluble PDGFR-β (PDGFR-β) monomeric and dimeric proteins (FIG.1A).

Two PDGFR-β monomeric constructs were made that contained a PDGFR-βsignal peptide (SP) at the N-terminus of the full length PDGFR-βectodomain, PDGFR(D1-D5) (SEQ ID NO:7), or at the N-terminus of aPDGFR-β ectodomain containing the first four ECDs, PDGFR(D1-D4) (SEQ IDNO:8). Three PDGFR-β dimeric constructs were produced by fusing allfive, first three, or first three domains of the PDGFR-β ectodomain(ECD) to the N-terminus of human immunoglobulin G1 heavy-chain fragment(IgG1 Fc) via a peptide linker consisting of nine glycine residues(9Gly) and were termed PDGFR(D1-D5)9G-Fc (SEQ ID NO:9),PDGFR(D1-D3)9G-Fc (SEQ ID NO:10) and PDGFR(D1-D2)9G-Fc (SEQ ID NO:11),respectively. Similar to the monomeric constructs, all dimericconstructs contained an SP at the N-terminus of the fused full-length ortruncated PDGFR-β ectodomains. For construction of PDGFR(D1-D2)9G-Fc,the plasmid pCMV6-XL5-PDGFRB (Cat.# SC309979; Origene, Rockville, Md.)was used as a template together with primers that introduced therestriction sites SpeI (PDGFRBPR6SpeI F: 5′-GACTAGTATGCGGCTTCCGGGTG (SEQID NO:25) and AgeI (PDGFRBPR7AgeI R: 5′-ACCGGTGGATGACACCTGGAGTCTG (SEQID NO:26) into the template. Amplification of the PCR products wasachieved with the following cycling parameters: 1 cycle at 50° C. for 2min, 1 cycle at 95° C. for 10 min; 40 cycles of 95° C. for 15 sec, and60° C. for 60 sec. The PCR product was inserted into pCR-Blunt II-TOPOplasmid using TOPO Cloning Kit (Invitrogen) and the sequence of the PCRproduct insert was verified by sequencing before subcloning into SpeIand AgeI sites of plasmid pCMV/K-D2-9Gly-Fc (See Pechan P., et al. GeneTher. (2009), 16:10-16 for a description of the pCMV/K-D2-9Gly-Fc) togenerate plasmid pCMV-PDGFR-S-(D1-D2)-9Gly-Fc with the open readingframe of PDGFR(D1-D2)9G-Fc (SEQ ID NO:20) under control of CMV promoterand SV40 polyadenylation sequence. For construction ofPDGFR(D1-D5)9G-Fc, the plasmid pCMV6-XL5-PDGFRB (Cat.# SC309979;Origene, Rockville, Md.) was used as a template together with primersthat introduced the restriction sites AccI (PDGFRB-PR1-Acc F:5′-CTATGTCTACAGACTCCAGGTGTC (SEQ ID NO:27) and AgeI (D5-PR9-AgeI-Rev R:5′-ACCGGTAAAGGGCAAGGAGTGTGGC (SEQ ID NO:28) into the template.Amplification of the PCR products was achieved with the followingcycling parameters: 1 cycle at 50° C. for 2 min, 1 cycle at 95° C. for10 min; 40 cycles of 95° C. for 15 sec, and 60° C. for 60 sec. The PCRproduct was then inserted into pCR-Blunt II-TOPO plasmid using TOPOCloning Kit (Invitrogen) and the sequence of PCR product insert in thepTOPO-PDGFRB (D3-D5) was verified by sequencing. The 622 base pair (bp)SpeI-AccI fragment from plasmid pCMV-PDGFR-S-(D1-D2)-9Gly-Fc wasinserted into SpeI and AccI sites of plasmid pTOPO-PDGFRB (D3-5) togenerate plasmid pTOPO-PDGFR(D1-D5). The 1,596 bp SpeI-AgeI fragmentfrom plasmid pTOPO-PDGFR(D1-D5) was then inserted into the SpeI and AgeIsites of plasmid pCMV-PDGFR-S-(D1-D2)-9Gly-Fc to generate the plasmidpCMV-PDGFR-(D1-D5)-9Gly-Fc with the open reading frame ofPDGFR(D1-D5)9G-Fc (SEQ ID NO:18) under control of the CMV promoter andSV40 polyadenylation sequence. For construction of PDGFR(D1-D3)9G-Fc,the plasmid pCMV-PDGFR (D1-5)9G-Fc was used as a template together withprimers that introduced the restriction sites SpeI (PDGF02 F:5′-CCTCCACCGGTGTAGCCGCTCTCAACCACGGT (SEQ ID NO:29) and AgeI (PDGF03 R:5′-CCCGGGACTAGTATGCGGCTTCCGGGTG (SEQ ID NO:30) into the template.Amplification of the PCR product was achieved with the following cyclingparameters: 1 cycle at 95° C. for 1 min; 35 cycles of 95° C. for 30 sec,60° C. for 30 sec, and 72° C. for 1 min. The PCR product was insertedinto the Spe I and Age I sites of plasmid pCMV-PDGFR (D1-5)9G-Fc tocomplete the open reading frame of PDGFR (D1-3)9G-Fc (SEQ ID NO:19). Forconstruction of PDGFR(D1-D5), the 5307 bp AgeI-EagI fragment of plasmidpCMV-sPDGFR(D1-D5)-9G-Fc was ligated to an annealed oligonucleotidefragment consisting of oligonucleotides D5-SV40 F: CCGGTTAGGGA (SEQ IDNO:31) and D5-SV40 B-2: GGCCTCCCTAA (SEQ ID NO:32) to generate plasmidpCMVPDGFRB (D1-D5) with the open reading frame of PDGFR(D1-D5) (SEQ IDNO:16) under control of the CMV promoter and SV40 polyadenylationsequence. For the construct PDGFR(D1-D4), a 4924 bp BbvCI-EagI fragmentof plasmid pCMV-sPDGFR(D1-D5)-9G-Fc was ligated to an annealedoligonucleotide fragment consisting of oligonucleotides D4-SV40 F:TGAGGTCCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGTA GC (SEQ IDNO:33) and D4-SV40 B:GGCCGCTACTCCAGCACTCGGACAGGGACATTGATCTGTAGCTGGAAGGAGAGCTG GACC (SEQ IDNO:34) to generate plasmid pCMV-PDGFRB (D1-D4) with the open readingframe of PDGFR(D1-D4) (SEQ ID NO:17) under control of the CMV promoterand SV40 polyadenylation sequence.

The predicted molecular weights for the mature proteins, excluding theSP region, was 56.2 kDa for PDGFR(D1-D5) and 43.6 kDa for PDGFR(D1-D4).The predicted molecular weights for the mature proteins as monomers,excluding the SP region, were 82.7 kDa for PDGFR(D1-D5)9G-Fc, 46.7 kDafor PDGFR(D1-D2)9G-Fc, and 58.2 kDa for PDGFR(D1-D3)9G-Fc. The plasmidsencoding these protein constructs were used for transfection of 293cells. Media from the cells was collected 72 hours post-transfection andcrude conditioned media (CM) was used for analysis of secretedPDGFR(D1-D5), PDGFR(D1-D4), PDGFR(D1-D5)9G-Fc, PDGFR(D1-D2)9G-Fc andPDGFR(D1-D3)9G-Fc proteins. Production of PDGFR(D1-D5)9G-Fc,PDGFR(D1-D2)9G-Fc and PDGFR(D1-D3)9G-Fc protein homodimers by cellstransfected with their respective constructs was confirmed by Westernblot analysis. Briefly, secreted proteins purified from cell culturemedia was loaded into reducing or non-reducing polyacrylamide gelelectrophoresis (PAGE) gels. Enhanced Green Fluorescent protein (EGFP)purified from cell culture media of cells transfected with an EGFPconstruct was also loaded on the gels and used as a control. Afterseparating the proteins on the SDS-PAGE gels (NuPAGE Novex 4-12%Bis-Tris, Invitrogen), the proteins were transferred to nitrocellulosemembranes. The membranes were probed with a biotinylated goat anti-humanPDGFR-β antibody (R&D Systems), followed by labeling with Streptavidinconjugated to horseradish peroxidase (R&D Systems) and developed withchemiluminescence reagent (ThermoScientific Pierce) prior to imaging.The mobility of PDGFR(D1-D5)9G-Fc, PDGFR(D1-D2)9G-Fc andPDGFR(D1-D3)9G-Fc proteins changed under reducing and non-reducingconditions while the mobility of the PDGFR(D1-D5) and PDGFR(D1-D4)monomer proteins remained unchanged indicating that the PDGFR-β/Fcfusion proteins formed homodimers (FIG. 1B).

The relative binding affinity between PDGF BB ligand and the PDGFR-βmonomeric and dimeric proteins was determined using a cell-freevolumetric PDGF binding assay system (FIG. 2). For production of PDGFR-βmonomeric and dimeric proteins, 293 cells were transfected with plasmidsencoding PDGFR(D1-D5), PDGFR(D1-D4), PDGFR(D1-D5)9G-Fc,PDGFR(D1-D2)9G-Fc, or PDGFR(D1-D3)9G-Fc proteins and cell culture mediawas harvested 72 hours post-transfection. The presence of secretedPDGFR-β monomeric and dimeric proteins was confirmed by ELISA andWestern blot analysis prior to binding affinity analysis. Secretedproteins were serially diluted, mixed with human PDGF BB ligand (20 pMfinal concentration) and incubated overnight at room temperature on anorbital shaker platform. The amount of unbound PDGF BB was then measuredby a human PDGF-specific ELISA (Human PDGF-BB DuoSet Product #DY220, R&DSystems). Statistical significance in binding affinities was analyzedusing Prism 5.0d (GraphPad Software, Inc) and was calculated using the2-way ANOVA test followed by Bonferroni correction. Binding affinityanalysis showed that monomeric PDGFR(D1-D4) protein bound PDGF withsignificantly (***P<0.001) higher affinity than monomeric PDGFR(D1-D5)protein that contained all 5 ECDs (FIG. 2A). However, the dimeric fulllength PDGFR(D1-D5)9G-Fc protein, that served as a positive control forPDGF binding was a significantly (***P<0.001) better PDGF binder thanboth monomeric PDGFR(D1-D4) and PDGFR(D1-D5) (FIG. 2A). Out of threedimeric IgG1 Fc-coupled PDGFR-β constructs generated, the construct withfirst three ECDs, PDGFR(D1-D3)9G-Fc, was a significantly (***P<0.001)better PDGF binder than the full-size PDGFR(D1-D5)9G-Fc protein, whilethe construct with the first two ECDs, PDGFR(D1-D2)9G-Fc showed noPDGF-binding affinity (FIG. 2B).

Example 2 Generation of Hybrid VEGFR1/PDGFR-β and PDGFR-β/VEGFR-1Proteins

The Flt-1 receptor (VEGFR-1) ectodomain contains 7 extracellular domains(ECD) numbered 1 to 7 from N-terminus to C-terminus of the protein. Inorder to block both PDGF BB and VEGF ligands, fusion proteins comprisingECDs of PDGFR-β and VEGFR1 were generated and termed hybrid proteins(FIG. 3).

A previously generated VEGF-binding protein, sFLT01, consisting of ECD 2of human VEGFR1 linked to a human immunoglobulin G1 heavy-chain fragment(IgG1 Fc) was used to generate DNA constructs encoding theVEGFR1/PDGFR-β or PDGFR-β/VEGFR1 hybrid proteins. See Pechan P., et al.Gene Ther. (2009), 16:10-16 for a description of the sFLT01 protein,which is incorporated herein by reference in its entirety.VEGFR1/PDGFR-β Hybrid 1 (SEQ ID NO:12) was constructed by linking afragment from sFLT01 containing the VEGFR1 signal peptide (SP) andVEGFR1 ECD 2 to the N-terminus of PDGFR-β ECD 1-5 via a peptide linkerconsisting of nine serine residues (9Ser) which was further linked toIgG 1 Fc via a peptide linker consisting of nine glycine residues(9Gly). PDGFR-β/VEGFR1 Hybrid 2 (SEQ ID NO:13) was constructed bylinking PDGFR-β ECD 1-5 and the PDGFR-β SP to the N-terminus of VEGFR1ECD 2 via a 9Ser peptide linker which was further linked to IgG1 Fc viaa 9Gly peptide linker. VEGFR1/PDGFR-β Hybrid 3 (SEQ ID NO:14) andPDGFR-β/VEGFR1 Hybrid 4 (SEQ ID NO:15) were composed similarly toHybrids 1 and 2, respectively, with the exception that PDGFR-β ECD 1-3was used instead of PDGFR-β ECD 1-5. For construction of VEGFRUPDGFR-βHybrid 1, a 372 bp SpeI-XhoI fragment Kozak-SP-D2-9Ser encoding a VEGFR1signal peptide fused to a VEGFR1 domain D2 comprising a 9-Serine (9Ser)linker (SEQ ID NO:35) was inserted into SpeI and XhoI sites of plasmidpTOPO-PDGFR(D1-D5) to generate plasmid pTOPO-(D2-9Ser)-PDGFR(D1-D5). TheSpeI-AgeI fragment from plasmid pTOPO-(D2-9Ser)-PDGFR(D1-D5) wasinserted into SpeI-AgeI sites of plasmid pCMV-PDGFR-(D1-D5)-9Gly-Fc tocreate pCMV-F(D2-95)-P(D1-D5)-9G-Fc or pCMV-Hybrid 1 with the openreading frame of VEGFRUPDGFR-β Hybrid 1 (SEQ ID NO:21) under control ofCMV promoter and SV40 polyadenylation sequence. For construction ofPDGFR-β/VEGFR1 Hybrid 2, a 678 bp BstBI-BmgXI fragment containingsynthetic DNA (GenScript) (SEQ ID NO:36) was ligated with 5626 bpBstBI-BmgXI fragment of plasmid pCMV-sPDGFR(D1-D5)-9G-Fc to createpCMV-P(D1-D5)-9S-F(D2)-9G-Fc or pCMV-Hybrid 2 with an open reading frameof PDGFR-β/VEGFR-1 Hybrid 2 (SEQ ID NO:22) under control of CMV promoterand SV40 polyadenylation sequence. For construction of VEGFRUPDGFR-βHybrid 3, a 452 bp BmgBI-PshAI fragment containing synthetic DNA(GenScript) (SEQ ID NO:37) was ligated with 5334 bp BmgBI-PshAI fragmentof plasmid pCMV-P(D1-D5)-9S-F(D2)-9G-Fc or pCMV-Hybrid 2 to createpCMV-F(D2-95)-P(D1-D3)-9G-Fc or pCMV-Hybrid 3 with open reading frame ofVEGFRUPDGFR-β Hybrid 3 (SEQ ID NO:23) under control of CMV promoter andSV40 polyadenylation sequence. For construction of PDGFR-β/VEGFR1 Hybrid4, a 758 bp BmgBI-PshAI fragment containing synthetic DNA (GenScript)(SEQ ID NO:38) was ligated with 5021 bp BmgBI-PshAI fragment of plasmidpCMV-P(D1-D5)-9S-F(D2)-9G-Fc or pCMV-Hybrid 2 to createpCMV-P(D1-D3)-9S-F(D2)-9G-Fc or pCMV-Hybrid 4 with open reading frame ofPDGFR-β/VEGFR1 Hybrid 4 (SEQ ID NO:24) under control of CMV promoter andSV40 polyadenylation sequence.

Production of Hybrid 1, Hybrid 2, Hybrid 3, and Hybrid 4 proteinhomodimers by cells transfected with their respective constructs wasconfirmed by Western blot analysis. Briefly, secreted proteins from cellculture media were loaded into reducing or non-reducing polyacrylamidegel electrophoresis (PAGE) gels. PDGFR(D1-D5)9G-Fc protein was alsoloaded on the gels and used as a control. After separating the proteinson the SDS-PAGE gels, (NuPAGE Novex 4-12% Bis-Tris, Invitrogen), theproteins were transferred to nitrocellulose membranes. The membraneswere probed with a biotinylated goat anti-human PDGFR-β antibody (R&DSystems), followed by labeling with Streptavidin conjugated tohorseradish peroxidase (R&D Systems) and developed withchemiluminescence reagent (ThermoScientific Pierce) prior to imaging.The protein mobility of Hybrids 1, 2, 3, and 4 under non-reducingconditions as compared to reducing conditions confirmed that the hybridproteins dimerized (FIG. 4). PDGFR(D1-D5)9G-Fc and Hybrids 1 and 2,which all contain five PDGFR-β ECDs, showed two PDGFR-positive bandsunder reducing conditions, suggesting a possible proteolytic cleavage ofthese proteins in the area of the fifth PDGFR-β ECD (FIG. 4, leftpanel). Hybrids 3 and 4, which contain only the first three PDGFR-βECDs, do not appear to be cleaved indicating that they do not containthe proteolytic cleavage site seen in Hybrids 1 and 2 (FIG. 4, leftpanel).

Example 3 Inhibition of HUVEC Proliferation by PDGFR-β/VEGFR1 HybridProteins

Hybrid PDGFR-β/VEGFR1 proteins were tested for their ability to inhibitVEGF- and/or PDGFR-β-induced proliferation of human umbilical veinendothelial cells (HUVECs). For production of hybrid proteins, 293 cellswere transfected with constructs encoding Hybrid 1, Hybrid 2, Hybrid 3,or Hybrid 4 and the cell culture media containing the secreted hybridproteins was harvested 72 hours after transfection. The harvested cellculture was applied to HUVECs in the presence of VEGF ligand. HUVECs(HUVEC-Cambrex Bio Science Walkersville, Inc) were seeded in a 96 wellplate at a density of 2,000 cells/well in Media 199 (Invitrogen)supplemented with 5% Fetal Bovine Serum (Invitrogen) and settledovernight. After incubation, the media was replaced with Media 199(Invitrogen) supplemented with 5% Fetal Bovine Serum (Invitrogen)containing an equal volume (50) of harvested cell culture generated fromthree independent receptor or control transfections together withrecombinant hVEGF-165 ligand alone at a final concentration of 10 ng/ml(R&D Systems Cat#293-VE), or in combination with PDGF-BB ligand at afinal concentration of 20 ng/ml (R&D Systems Cat#220-BB) in a finalvolume of 100 μl per well. Negative controls consisted of an equalvolume (50) of harvested cell culture from cells transfected with anEGFP construct at a 100 μL final volume per well. Positive controlsincluded harvested cell culture of an equal volume (50) from cellstransfected with an EGFP construct in the presence of VEGF ligand orVEGF and PDGF BB ligand 100 μL final volume per well. Cells wereincubated at 37° C. in 5% CO₂ for three to four days. Cell Titer 96AQ_(ueous) One Solution Reagent (Promega Cat# G3580) was added at 20μl/well and absorbance at 490 nm was taken four hours later. In theVEGF-dependent HUVEC proliferation assay, Hybrid 2, Hybrid 3, and Hybrid4 significantly blocked HUVEC proliferation with Hybrids 2 and 4 havinga similar potency as sFLT01 (FIG. 5A). In comparison to Hybrids 2, 3,and 4, Hybrid 1 did not block VEGF-induced HUVEC proliferation and hadsimilar levels of anti-proliferative activity as PDGFR(D1-D5)9G-Fcprotein that lacks VEGFR1 ECDs (FIG. 5A). The proteolytic excision ofthe dimerizing IgG1-Fc sequence in Hybrid 1 probably eliminated its VEGFbinding ability, because dimerization is a limiting factor for VEGFR1 D2mediated VEGF binding when other ECDs are not present (Pechan P., et al.Gene Ther. (2009), 16:10-16). In Hybrid 2, however, proteolytic cleavageseparated the molecule into PDGFR-β ECDs and sFLT01-containing unitsthat were still able to bind VEGF (FIG. 5A). The harvested hybridPDGFR-β/VEGFR1 proteins were also tested in a HUVEC competitiveproliferation assay. In this assay, harvested cell culture was appliedto HUVECs in the presence of both VEGF ligand and PDGF BB ligand asdescribed above. Results from this assay were similar to the previousassay in that Hybrid 2 and Hybrid 4 significantly blocked HUVECproliferation with Hybrids 2 and 4 having a similar potency as sFLT01(FIG. 5B). Hybrid 1 also did not block HUVEC proliferation in thepresence of both ligands. In contrast to the last assay, Hybrid 3 hadweaker ant-proliferative potency in the presence of both ligands and wascomparable to activity of the PDGFR(D1-D5)9G-Fc protein that lacksVEGFR1 ECDs (FIG. 5B). Statistical significance for both HUVECproliferation assays was analyzed using Prism 5.0d (GraphPadSoftwareInc) and calculated using the one-way ANOVA test followed by Tukey'sTest.

Example 4 Binding Affinities for PDGFR-β/VEGFR1 Hybrid Proteins

The relative binding affinities between both VEGF and PDGF BB ligandsand the PDGFR-β/VEGFR1 hybrid proteins was determined using a cell-freevolumetric PDGF or VEGF binding assay system (FIG. 6). For production ofhybrid PDGFR-β/VEGFR1 proteins, 293 cells were transfected with plasmidsencoding Hybrid 1, Hybrid 2, Hybrid 3, or Hybrid 4. Cells were alsotransfected with plasmids encoding PDGFR(D1-D5)9G-Fc or sFLT01 proteinsfor use as binding controls. Cell culture media was harvested 72 hourspost-transfection and the presence of secreted proteins was confirmed byELISA and Western blot analysis prior to binding affinity analysis.Secreted proteins were serially diluted, mixed with human VEGFR1 ligand(20 pM final concentration) or human PDGF BB ligand (80 pm finalconcentration) and incubated overnight at room temperature on an orbitalshaker platform. The amount of unbound PDGF BB was then measured by ahuman VEGF-specific ELISA (Human VEGF Quantikine ELISA kit Cat# DVE00,R&D Systems) or a human PDGF-specific ELISA (Human PDGF-BB DuoSet, R&DSystems). Comparison of all four hybrids in the VEGF binding assayshowed that Hybrid 1 was the weakest VEGF binder (FIG. 6A). VEGF bindingcomparison of Hybrid 3, Hybrid 4 and sFLT01 in conditioned mediaharvested from three individual transfections in one assay showed thatHybrid 4 bound VEGF similarly to sFLT01 and was a stronger VEGF binderthan Hybrid 3 (FIG. 6A). Comparison of all four hybrids in the PDGFbinding assay demonstrated that Hybrid 1 was also the weakest PDGFbinder while Hybrid 4 demonstrated the best binding out of all fourhybrids (FIG. 6B).

Competitive VEGF and PDGF cell-free binding assays were conducted usingconditioned media harvested from 293 cells transfected with constructsencoding Hybrid 3, Hybrid 4, PDGFR(D1-D3)9G-Fc, or sFLT01 proteins. Cellculture media was harvested 72 hours post-transfection and the presenceof secreted proteins was confirmed by ELISA and Western blot analysisprior to binding affinity analysis. Secreted proteins were seriallydiluted, mixed with both human PDGF BB ligand (20 pM finalconcentration) and human VEGFR1 ligand (20 pM final concentration) andincubated overnight at room temperature on an orbital shaker platform.The amount of unbound PDGF-BB and VEGF ligands was subsequently measuredby a human VEGF-specific ELISA (Human VEGF Quantikine ELISA kit Cat#DVE00, R&D Systems) or a human PDGF-specific ELISA (Human PDGF-BBDuoSet, R&D Systems). Comparison of Hybrid 3 and Hybrid 4 to the PDGF BBbinding control (PDGFR(D1-D3)9G-Fc) and VEGF binding control (sFLT01)showed that Hybrid 3 and Hybrid 4 both bound to PDGF BB (FIG. 7A) andVEGF (FIG. 7B) ligands, with Hybrid 4 demonstrating a higher bindingaffinity to both ligands. PDGF binding comparison using cell culturemedia from cells expressing Hybrid 3, Hybrid 4, PDGFR(D1-D5)9G-Fc, orPDGFR(D1-D3)9G-Fc demonstrated that Hybrid 4 had a similar affinity tothe best PDGF binder, PDGFR(D1-D3)9G-Fc (FIG. 7A) whilePDGFR(D1-D5)9G-Fc was a significantly weaker PDGF binder than all ofHybrids 1 to 4 (FIG. 6B) or PDGFR(D1-D3)9G-Fc (FIG. 2B).

Example 5 Inhibition of Laser-Induced CNV in Mice by HybridPDGFR-β/VEGFR1 Proteins

Adeno-associated virus (AAV) vectors are attractive tools forintraocular gene delivery because of their nonpathogenic nature, minimaltoxicity and immunogenicity, their ability to transduce nondividingcells, and their potential for a life-time expression of a therapeuticprotein (Ali et al. 1996; Ali et al. 1997; Ali et al., 1998; Lai et al.2005).

For adeno-associated virus-mediated delivery of Hybrid 4, the CMVpromoter was replaced by a chicken beta-actin promoter-CMVintron/enhancer and the an expression cassette comprising the promoterand the fragment encoding Hybrid 4 was inserted into the RsrII and MluIsites of a previral plasmid vector pAAV5P70. See Ziegler et al. Mol.Ther., 2004; 9: 231-240. The total size of the resulting AAV genome inplasmid sp70.BR/Hybrid 4 was 4.6 kb. The recombinant vector AAV2.Hybrid4 was produced by triple transfection of 293 cells using helper plasmidsp5rep-D-CMVcap and pHelper (Stratagene, La Jolla, Calif., USA), andpurified according to the manufacture's protocol using an iodixanol stepgradient and HiTrap Heparin column (GE Healthcare Life Sciences,Piscataway, N.J., USA) on an A “KTA FPLC system (GE Healthcare LifeSciences, Piscataway, N.J.). See Vincent at al., J. Virol., 1997; 71:1897-1905. The AAV2.Hybrid 4 viral preparation had a titer of 2.2E12drps (DNase resistant particles) per ml. Viral titers were determinedusing a real-time TaqMan PCR assay (ABI Prism 7700; Applied Biosystems,Foster City, Calif., USA). AAV2.sFLT02 was constructed as previouslydescribed in U.S. Pat. No. 7,928,072 using a nucleic acid (SEQ ID NO:40)encoding for the VEGFR1 D2-9Gly-CH3 protein (SEQ ID NO:39). AAV2.Hybrid4, AAV2.sFLT02, or AAV2.PDGFR (PDGFR=PDGFR(D1-D3)9G-Fc) was administeredby intravitreal delivery in a mouse choroidal neovascularization (CNV)laser model to assess the in vivo efficacy of Hybrid 4 in the inhibitionof CNV. Briefly, the eyes of normal adult C57BL/6 mice were treated witha single intravitreal injection of 1 E9 drps of AAV2.Hybrid 4,AAV2.sFLT02, or AAV2.PDGFR into the left eye (OS) on study day 0 whilethe right eye (OD) was left naïve to treatment. CNV was induced in botheyes using a laser (3 burns placed per eye. 200 mW power, 50 μm spot,100 ms) on study day 28. Mice were perfused with 5 mg/mL of 2.0×10⁶molecular weight FITC-Dextran and euthanized on study day 42. The eyeswere collected, fixed in 10% neutral buffered formalin and choroidalflatmounts were subsequently prepared in order to examine the extent ofneovascularization. The number of burns without CNV in the treated (OS)eye was compared to the contralateral (OD) eye. Analysis of in vivoefficacy demonstrated that a single intravitreal injection ofAAV2.Hybrid 4 was more effective than AAV2.sFLT02 in the inhibition ofretinal neovascularization (FIG. 8). Furthermore, AAV2.PDGFR did notinhibit retinal neovascularization in the murine laser-induced CNV model(FIG. 8).

SEQUENCES

PDGFR extracellular region D1-D3 amino acid sequence (SEQ ID NO: 1)LVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYPDGFR extracellular region D1-D4 amino acid sequence (SEQ ID NO: 2)LVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLEPDGFR extracellular region D1-D5 amino acid sequence (SEQ ID NO: 3)LVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHVDRPLSVRCTLRNAVGQDTQEVIVVPHSLPFK VEGFR1 extracellular region D2 amino acid sequence(SEQ ID NO: 4)RPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTVEGFR1 extracellular region D1-D3 amino acid sequence (SEQ ID NO: 5)PELSLKGTQHIMQAGQTLHLQCRGEAAHKWSLPEMVSKESERLSITKSACGRNGKQFCSTLTLNTAQANHTGFYSCKYLAVPTSKKKETESAIYIFISDTGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTNTIIDVQISTPRPVKLLRGHTLVLNCTATTPLNTRVQMTWSYPDEKNKRASVRRRIDQSNSHANIFYSVLTIDKMQNKDKGLYTCRVRSGPSFKSVNTSVHIYDKIgG1 Fc region amino acid sequence (SEQ ID NO: 6)PKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPDGFR(D1-D5) amino acid sequence with secretory peptide (underlined)(SEQ ID NO: 7)MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHVDRPLSVRCTLRNAVGQDTQEVIVVPHSLPFKPDGFR(D1-D4) amino acid sequence with secretory peptide (underlined)(SEQ ID NO: 8)MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVR VLEPDGFR(D1-D5)9G-Fc amino acid sequence with secretory peptide (underlined)(SEQ ID NO: 9)MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHVDRPLSVRCTLRNAVGQDTQEVIVVPHSLPFKGGGGGGGGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KPDGFR(D1-D3)9G-Fc amino acid sequence with secretory peptide (underlined)(SEQ ID NO: 10)MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYGGGGGGGGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKPDGFR(D1-D2)9G-Fc amino acid sequence with secretory peptide (underlined)(SEQ ID NO: 11)MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSGGGGGGGGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Hybrid 1 amino acid sequence(SEQ ID NO: 12)MVSYWDTGVLLCALLSCLLLTGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTSSSSSSSSSQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHVDRPLSVRCTLRNAVGQDTQEVIVVPHSLPFTGGGGGGGGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Hybrid 2 amino acid sequence(SEQ ID NO: 13)MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYVRLLGEVGTLQFAELHRSRTLQVVFEAYPPPTVLWFKDNRTLGDSSAGEIALSTRNVSETRYVSELTLVRVKVAEAGHYTMRAFHEDAEVQLSFQLQINVPVRVLELSESHPDSGEQTVRCRGRGMPQPNIIWSACRDLKRCPRELPPTLLGNSSEEESQLETNVTYWEEEQEFEVVSTLRLQHVDRPLSVRCTLRNAVGQDTQEVIVVPHSLPFSSSSSSSSSRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTGGGGGGGGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK Hybrid 3 amino acid sequence(SEQ ID NO: 14)MVSYWDTGVLLCALLSCLLLTGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTSSSSSSSSSQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYTGGGGGGGGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKHybrid 4 amino acid sequence (SEQ ID NO: 15)MRLPGAMPALALKGELLLLSLLLLLEPQISQGLVVTPPGPELVLNVSSTFVLTCSGSAPVVWERMSQEPPQEMAKAQDGTFSSVLTLTNLTGLDTGEYFCTHNDSRGLETDERKRLYIFVPDPTVGFLPNDAEELFIFLTEITEITIPCRVTDPQLVVTLHEKKGDVALPVPYDHQRGFFGIFEDRSYICKTTIGDREVDSDAYYVYRLQVSSINVSVNAVQTVVRQGENITLMCIVIGNEVVNFEWTYPRKESGRLVEPVTDFLLDMPYHIRSILHIPSAELEDSGTYTCNVTESVNDHQDEKAINITVVESGYSSSSSSSSSRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTGGGGGGGGGPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG KPDGFR(D1-D5) open reading frame (SEQ ID NO: 16)ATGCGGCTTCCGGGTGCGATGCCAGCTCTGGCCCTCAAAGGCGAGCTGCTGTTGCTGTCTCTCCTGTTACTTCTGGAACCACAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACGTGCGGCTCCTGGGAGAGGTGGGCACACTACAATTTGCTGAGCTGCATCGGAGCCGGACACTGCAGGTAGTGTTCGAGGCCTACCCACCGCCCACTGTCCTGTGGTTCAAAGACAACCGCACCCTGGGCGACTCCAGCGCTGGCGAAATCGCCCTGTCCACGCGCAACGTGTCGGAGACCCGGTATGTGTCAGAGCTGACACTGGTTCGCGTGAAGGTGGCAGAGGCTGGCCACTACACCATGCGGGCCTTCCATGAGGATGCTGAGGTCCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGCTAAGTGAGAGCCACCCTGACAGTGGGGAACAGACAGTCCGCTGTCGTGGCCGGGGCATGCCCCAGCCGAACATCATCTGGTCTGCCTGCAGAGACCTCAAAAGGTGTCCACGTGAGCTGCCGCCCACGCTGCTGGGGAACAGTTCCGAAGAGGAGAGCCAGCTGGAGACTAACGTGACGTACTGGGAGGAGGAGCAGGAGTTTGAGGTGGTGAGCACACTGCGTCTGCAGCACGTGGATCGGCCACTGTCGGTGCGCTGCACGCTGCGCAACGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCT TGCCCTTTTAAPDGFR(D1-D4) open reading frame (SEQ ID NO: 17)ATGCGGCTTCCGGGTGCGATGCCAGCTCTGGCCCTCAAAGGCGAGCTGCTGTTGCTGTCTCTCCTGTTACTTCTGGAACCACAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACGTGCGGCTCCTGGGAGAGGTGGGCACACTACAATTTGCTGAGCTGCATCGGAGCCGGACACTGCAGGTAGTGTTCGAGGCCTACCCACCGCCCACTGTCCTGTGGTTCAAAGACAACCGCACCCTGGGCGACTCCAGCGCTGGCGAAATCGCCCTGTCCACGCGCAACGTGTCGGAGACCCGGTATGTGTCAGAGCTGACACTGGTTCGCGTGAAGGTGGCAGAGGCTGGCCACTACACCATGCGGGCCTTCCATGAGGATGCTGAGGTCCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGTAG PDGFR(D1-D5)9G-Fc open reading frame(SEQ ID NO: 18)ATGCGGCTTCCGGGTGCGATGCCAGCTCTGGCCCTCAAAGGCGAGCTGCTGTTGCTGTCTCTCCTGTTACTTCTGGAACCACAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACGTGCGGCTCCTGGGAGAGGTGGGCACACTACAATTTGCTGAGCTGCATCGGAGCCGGACACTGCAGGTAGTGTTCGAGGCCTACCCACCGCCCACTGTCCTGTGGTTCAAAGACAACCGCACCCTGGGCGACTCCAGCGCTGGCGAAATCGCCCTGTCCACGCGCAACGTGTCGGAGACCCGGTATGTGTCAGAGCTGACACTGGTTCGCGTGAAGGTGGCAGAGGCTGGCCACTACACCATGCGGGCCTTCCATGAGGATGCTGAGGTCCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGCTAAGTGAGAGCCACCCTGACAGTGGGGAACAGACAGTCCGCTGTCGTGGCCGGGGCATGCCCCAGCCGAACATCATCTGGTCTGCCTGCAGAGACCTCAAAAGGTGTCCACGTGAGCTGCCGCCCACGCTGCTGGGGAACAGTTCCGAAGAGGAGAGCCAGCTGGAGACTAACGTGACGTACTGGGAGGAGGAGCAGGAGTTTGAGGTGGTGAGCACACTGCGTCTGCAGCACGTGGATCGGCCACTGTCGGTGCGCTGCACGCTGCGCAACGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAAT AGPDGFR(D1-D3)9G-Fc open reading frame (SEQ ID NO: 19)ATGCGGCTTCCGGGTGCGATGCCAGCTCTGGCCCTCAAAGGCGAGCTGCTGTTGCTGTCTCTCCTGTTACTTCTGGAACCACAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG PDGFR(D1-D2)9G-Fc open reading frame(SEQ ID NO: 20)ATGCGGCTTCCGGGTGCGATGCCAGCTCTGGCCCTCAAAGGCGAGCTGCTGTTGCTGTCTCTCCTGTTACTTCTGGAACCACAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCG GGTAAATAGHybrid 1 open reading frame (SEQ ID NO: 21)ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTGTCTGCTTCTCACAGGATCTGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCTCGAGTTCCAGCTCCTCTTCCTCAAGCCAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACGTGCGGCTCCTGGGAGAGGTGGGCACACTACAATTTGCTGAGCTGCATCGGAGCCGGACACTGCAGGTAGTGTTCGAGGCCTACCCACCGCCCACTGTCCTGTGGTTCAAAGACAACCGCACCCTGGGCGACTCCAGCGCTGGCGAAATCGCCCTGTCCACGCGCAACGTGTCGGAGACCCGGTATGTGTCAGAGCTGACACTGGTTCGCGTGAAGGTGGCAGAGGCTGGCCACTACACCATGCGGGCCTTCCATGAGGATGCTGAGGTCCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGCTAAGTGAGAGCCACCCTGACAGTGGGGAACAGACAGTCCGCTGTCGTGGCCGGGGCATGCCCCAGCCGAACATCATCTGGTCTGCCTGCAGAGACCTCAAAAGGTGTCCACGTGAGCTGCCGCCCACGCTGCTGGGGAACAGTTCCGAAGAGGAGAGCCAGCTGGAGACTAACGTGACGTACTGGGAGGAGGAGCAGGAGTTTGAGGTGGTGAGCACACTGCGTCTGCAGCACGTGGATCGGCCACTGTCGGTGCGCTGCACGCTGCGCAACGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG Hybrid 2 open reading frame (SEQ ID NO: 22)ATGCGGCTTCCGGGTGCGATGCCAGCTCTGGCCCTCAAAGGCGAGCTGCTGTTGCTGTCTCTCCTGTTACTTCTGGAACCACAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACGTGCGGCTCCTGGGAGAGGTGGGCACACTACAATTTGCTGAGCTGCATCGGAGCCGGACACTGCAGGTAGTGTTCGAGGCCTACCCACCGCCCACTGTCCTGTGGTTCAAAGACAACCGCACCCTGGGCGACTCCAGCGCTGGCGAAATCGCCCTGTCCACGCGCAACGTGTCGGAGACCCGGTATGTGTCAGAGCTGACACTGGTTCGCGTGAAGGTGGCAGAGGCTGGCCACTACACCATGCGGGCCTTCCATGAGGATGCTGAGGTCCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGCTAAGTGAGAGCCACCCTGACAGTGGGGAACAGACAGTCCGCTGTCGTGGCCGGGGCATGCCCCAGCCGAACATCATCTGGTCTGCCTGCAGAGACCTCAAAAGGTGTCCACGTGAGCTGCCGCCCACGCTGCTGGGGAACAGTTCCGAAGAGGAGAGCCAGCTGGAGACTAACGTGACGTACTGGGAGGAGGAGCAGGAGTTTGAGGTGGTGAGCACACTGCGTCTGCAGCACGTGGATCGGCCACTGTCGGTGCGCTGCACGCTGCGCAACGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAGTTCCAGCTCCTCTTCCTCAAGCTCGCCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG Hybrid 3 open reading frame (SEQ ID NO: 23)ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTGTCTGCTTCTCACAGGATCTGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCTCGAGTTCCAGCTCCTCTTCCTCAAGCCAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAGHybrid 4 open reading frame (SEQ ID NO: 24)ATGCGGCTTCCGGGTGCGATGCCAGCTCTGGCCCTCAAAGGCGAGCTGCTGTTGCTGTCTCTCCTGTTACTTCTGGAACCACAGATCTCTCAGGGCCTGGTCGTCACACCCCCGGGGCCAGAGCTTGTCCTCAATGTCTCCAGCACCTTCGTTCTGACCTGCTCGGGTTCAGCTCCGGTGGTGTGGGAACGGATGTCCCAGGAGCCCCCACAGGAAATGGCCAAGGCCCAGGATGGCACCTTCTCCAGCGTGCTCACACTGACCAACCTCACTGGGCTAGACACGGGAGAATACTTTTGCACCCACAATGACTCCCGTGGACTGGAGACCGATGAGCGGAAACGGCTCTACATCTTTGTGCCAGATCCCACCGTGGGCTTCCTCCCTAATGATGCCGAGGAACTATTCATCTTTCTCACGGAAATAACTGAGATCACCATTCCATGCCGAGTAACAGACCCACAGCTGGTGGTGACACTGCACGAGAAGAAAGGGGACGTTGCACTGCCTGTCCCCTATGATCACCAACGTGGCTTTTTTGGTATCTTTGAGGACAGAAGCTACATCTGCAAAACCACCATTGGGGACAGGGAGGTGGATTCTGATGCCTACTATGTCTACAGACTCCAGGTGTCATCCATCAACGTCTCTGTGAACGCAGTGCAGACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACAGTTCCAGCTCCTCTTCCTCAAGCTCGAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTGAGCCACGAAGACCCTGAGGTCAAGTTCAACTGGTACGTGGACGGCGTGGAGGTGCATAATGCCAAGACAAAGCCGCGGGAGGAGCAGTACAACAGCACGTACCGTGTGGTCAGCGTCCTCACCGTCCTGCACCAGGACTGGCTGAATGGCAAGGAGTACAAGTGCAAGGTCTCCAACAAAGCCCTCCCAGCCCCCATCGAGAAAACCATCTCCAAAGCCAAAGGGCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAGPDGFRBPR6SpeI F nucleic acid primer (SEQ ID NO: 25)GACTAGTATGCGGCTTCCGGGTG PDGFRBPR7AgeI R nucleic acid primer(SEQ ID NO: 26) ACCGGTGGATGACACCTGGAGTCTGPDGFRB-PR1-Acc F nucleic acid primer (SEQ ID NO: 27)CTATGTCTACAGACTCCAGGTGTC D5-PR9-AgeI-Rev R nucleic acid primer(SEQ ID NO: 28) ACCGGTAAAGGGCAAGGAGTGTGGC PDGF02 F nucleic acid primer(SEQ ID NO: 29) CCTCCACCGGTGTAGCCGCTCTCAACCACGGTPDGF03 R nucleic acid primer (SEQ ID NO: 30)CCCGGGACTAGTATGCGGCTTCCGGGTG D5-SV40 F nucleic acid primer(SEQ ID NO: 31) CCGGTTAGGGA D5-SV40 B-2 nucleic acid primer(SEQ ID NO: 32) GGCCTCCCTAA D4-SV40 F nucleic acid primer(SEQ ID NO: 33)TGAGGTCCAGCTCTCCTTCCAGCTACAGATCAATGTCCCTGTCCGAGTGCTGGAGTA GCD4-SV40 B nucleic acid primer (SEQ ID NO: 34)GGCCGCTACTCCAGCACTCGGACAGGGACATTGATCTGTAGCTGGAAGGAGAGCTG GACCKozak-SP-D2-9Ser synthetic nucleic acid fragment (SEQ ID NO: 35)ACTAGTGGCGGCCGCCACCATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTGTCTGCTTCTCACAGGATCTGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCTCGAGTTCCAGCTCCTCTTCCTCA AGCCAGATCTD2-2220-2908 synthetic nucleic acid fragment (SEQ ID NO: 36)CCACGCTGCTGGGGAACAGTTCCGAAGAGGAGAGCCAGCTGGAGACTAACGTGACGTACTGGGAGGAGGAGCAGGAGTTTGAGGTGGTGAGCACACTGCGTCTGCAGCACGTGGATCGGCCACTGTCGGTGCGCTGCACGCTGCGCAACGCTGTGGGCCAGGACACGCAGGAGGTCATCGTGGTGCCACACTCCTTGCCCTTTAGTTCCAGCTCCTCTTCCTCAAGCTCGAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGT GGTGGACGTGD3-Fc synthetic nucleic acid fragment (SEQ ID NO: 37)GACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGG TGGACGTGD3-F(D2) synthetic nucleic acid fragment (SEQ ID NO: 38)GACTGTGGTCCGCCAGGGTGAGAACATCACCCTCATGTGCATTGTGATCGGGAATGAGGTGGTCAACTTCGAGTGGACATACCCCCGCAAAGAAAGTGGGCGGCTGGTGGAGCCGGTGACTGACTTCCTCTTGGATATGCCTTACCACATCCGCTCCATCCTGCACATCCCCAGTGCCGAGTTAGAAGACTCGGGGACCTACACCTGCAATGTGACGGAGAGTGTGAATGACCATCAGGATGAAAAGGCCATCAACATCACCGTGGTTGAGAGCGGCTACAGTTCCAGCTCCTCTTCCTCAAGCTCGAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCCTAAATCTTGTGACAAAACTCACACATGCCCACCGTGCCCAGCACCTGAACTCCTGGGGGGACCGTCAGTCTTCCTCTTCCCCCCAAAACCCAAGGACACCCTCATGATCTCCCGGACCCCTGAGGTCACATGCGTGGTGGTGGACGTG sFLT02 (D2-9Gly-CH3) amino acid sequence(SEQ ID NO: 39)MVSYWDTGVLLCALLSCLLLTGSGRPFVEMYSEIPEIIHMTEGRELVIPCRVTSPNITVTLKKFPLDTLIPDGKRIIWDSRKGFIISNATYKEIGLLTCEATVNGHLYKTNYLTHRQTGGGGGGGGGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKsFLT02 (D2-9Gly-CH3) open reading frame (SEQ ID NO: 40)ATGGTCAGCTACTGGGACACCGGGGTCCTGCTGTGCGCGCTGCTCAGCTGTCTGCTTCTCACAGGATCTGGTAGACCTTTCGTAGAGATGTACAGTGAAATCCCCGAAATTATACACATGACTGAAGGAAGGGAGCTCGTCATTCCCTGCCGGGTTACGTCACCTAACATCACTGTTACTTTAAAAAAGTTTCCACTTGACACTTTGATCCCTGATGGAAAACGCATAATCTGGGACAGTAGAAAGGGCTTCATCATATCAAATGCAACGTACAAAGAAATAGGGCTTCTGACCTGTGAAGCAACAGTCAATGGGCATTTGTATAAGACAAACTATCTCACACATCGACAAACCGGTGGAGGTGGAGGTGGAGGTGGAGGTCAGCCCCGAGAACCACAGGTGTACACCCTGCCCCCATCCCGGGATGAGCTGACCAAGAACCAGGTCAGCCTGACCTGCCTGGTCAAAGGCTTCTATCCCAGCGACATCGCCGTGGAGTGGGAGAGCAATGGGCAGCCGGAGAACAACTACAAGACCACGCCTCCCGTGCTGGACTCCGACGGCTCCTTCTTCCTCTACAGCAAGCTCACCGTGGACAAGAGCAGGTGGCAGCAGGGGAACGTCTTCTCATGCTCCGTGATGCATGAGGCTCTGCACAACCACTACACGCAGAAGAGCCTCTCCCTGTCTCCGGGTAAATAG

1. A fusion protein comprising (a) an extracellular portion of a PDGFreceptor, (b) an extracellular portion of a VEGF receptor, and (c) amultimerization domain, wherein the fusion proteins binds to a PDGF anda VEGF, and the fusion protein is arranged from N-terminus to C-terminusin the following order: (a), (b) and (c).
 2. The fusion protein of claim1, wherein the PDGF receptor is a PDGFRβ.
 3. The fusion protein of claim1, wherein the extracellular portion of the PDGFR comprises: (a) theIg-like domains D1-D3 of the PDGFR; (b) the Ig-like domains D1-D4 of thePDGFR; (c) the Ig-like domains D1-D5 of the PDGFR; (d) the amino acidsequence SEQ ID NO:1, 2, or 3; or (e) an amino acid sequence having atleast 85% identity to SEQ ID NO:1, 2, or
 3. 4-6. (canceled)
 7. Thefusion protein of claim 1, wherein the extracellular portion of the VEGFreceptor comprises: (a) an Ig-like domain D2 of a VEGF receptor; (b) anIg-like domain D2 of a VEGFR1 (FLT-1); (c) an Ig-like domain D2 of aVEGFR1 (FLT-1) and an Ig-like domain D3 of a VEGFR2; (d) Ig-like domainsD1-D3 of a VEGFR1 (FLT-1); (e) the amino acid sequence of SEQ ID NO:4 or5; or (f) an amino acid sequence having at least 85% identity to SEQ IDNO:4 or
 5. 8-11. (canceled)
 12. The fusion protein of claim 1, whereinthe fusion protein further comprises a linker peptide between theextracellular portion of the PDGF receptor and the extracellular portionof the VEGF receptor, and/or a peptide linker between the extracellularportion of the VEGF receptor and the multimerization domain.
 13. Thefusion protein of claim 12, wherein the peptide linker comprises theamino acid sequence selected from the group consisting of Gly₉ (SEQ IDNO:42), Glu₉ (SEQ ID NO:43), Ser₉ (SEQ ID NO:44), Gly₅ Cys-Pro₂-Cys (SEQID NO:45), (Gly₄-Ser)₃ (SEQ ID NO:46),Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn (SEQ ID NO:47),Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn (SEQ ID NO:48),Gly-Asp-Leu-Ile-Tyr-Arg-Asn-Gln-Lys (SEQ ID NO:49), andGly₉-Pro-Ser-Cys-Val-Pro-Leu-Met-Arg-Cys-Gly-Gly-Cys-Cys-Asn (SEQ IDNO:50).
 14. The fusion protein of claim 1, wherein the multimerizationdomain is: (a) a Fc region of an antibody; (b) a Fc region of anantibody comprising a CH3 region of IgG1, IgG2, IgG3, or IgG4, or a CH2and a CH3 region of IgG1, IgG2, IgG3, or IgG4; (c) a Fc region of anantibody comprising the amino acid sequence of SEQ ID NO:6; or (d) a Fcregion of an antibody comprising an amino acid sequence having at least85% identity to SEQ ID NO:6. 15-16. (canceled)
 17. The fusion protein ofclaim 1, wherein the fusion protein comprises the amino acid sequence ofSEQ ID NO:13 or 15, or an amino acid sequence having at least 85%identity to SEQ ID NO:13 or
 15. 18. The fusion protein of claim 1,wherein the fusion protein is in a dimeric or a multimeric form. 19-21.(canceled)
 22. A composition comprising the fusion protein claim 1 and apharmaceutically acceptable carrier.
 23. A nucleic acid encoding thefusion protein of claim
 1. 24. A host cell comprising a nucleotidesequence encoding the fusion protein of claim
 1. 25. A method ofproducing a fusion protein, comprising culturing a host cell comprisinga nucleic acid encoding the fusion protein of claim 1 under a conditionthat produces the fusion protein, and recovering the fusion proteinproduced by the host cell.
 26. (canceled)
 27. A method of delivering afusion protein to a subject comprising administering an effective amountof the fusion protein of claim 1 to the subject.
 28. The method of claim27, wherein the subject has macular degeneration or proliferativediabetic retinopathy, cancer, rheumatoid arthritis, osteoarthritis,asthma, uveitis or corneal neovascularization. 29-33. (canceled)
 34. Avector comprising a nucleotide sequence encoding the fusion protein ofclaim
 1. 35-37. (canceled)
 38. An rAAV particle comprising a nucleicacid encoding the fusion protein of claim
 1. 39-41. (canceled)
 42. Amethod of producing an rAAV particle, comprising: (a) culturing a hostcell under a condition that rAAV particles are produced, wherein thehost cell comprises (i) one or more AAV package genes, wherein each saidAAV packaging gene encodes an AAV replication or encapsidation protein;(ii) an rAAV pro-vector comprising a nucleotide encoding the fusionprotein of claim 1 flanked by at least one AAV ITR, and (iii) an AAVhelper function; and (b) recovering the rAAV particles produced by thehost cell.
 43. (canceled)
 44. A method of delivering a viral vector to asubject, comprising administering the rAAV particle of claim 38 to thesubject, wherein the fusion protein encoded by the rAAV particle isexpressed in the subject.
 45. The method of claim 44, wherein thesubject has macular degeneration, proliferative diabetic retinopathy,rheumatoid arthritis, osteoarthritis, asthma, uveitis or cornealneovascularization. 46-50. (canceled)